Compositions and methods for treatment of neoplastic disease

ABSTRACT

The present invention comprises the use of sickle cells or sickle cell precursors loaded with a therapeutic agent that localize in tumors and induce a tumoricidal response.

CROSS REFERENCE TO RELATED APPLICATIONS

The instant application is a divisional of U.S. application Ser. No.10/428,817, filed on May 5, 2003, which is a continuation in part ofdivisional 12/145,949, filed on Jun. 25, 2008, which is a continuationof U.S. application Ser. No. 10/937,758, filed on Sep. 8, 2004, which isa continuation of U.S. application Ser. No. 09/650,884, filed on Aug.30, 2000, which claims priority to provisional application 60/151,470,filed on Aug. 30, 1999. All of the above referenced applications areincorporated in their entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to therapeutic compositions and methodsfor treating tumors and cancer.

2. Description of the Background Art

Therapy of the neoplastic diseases has largely involved the use ofchemotherapeutic agents, radiation, and surgery. However, results withthese measures, while beneficial in some tumors, has had only marginaleffects in many patients and little or no effect in many others, whiledemonstrating unacceptable toxicity. Hence, there has been a quest fornewer modalities to treat neoplastic diseases.

Erythrocytes from patients with sickle cell anemia contain a highpercentage of SS hemoglobin which under conditions of deoxygenationaggregate followed by the growth and alignment of fibers transformingthe cell into a classic sickle shape. Retardation of the transit time ofsickled erythrocytes results in vaso-occlusion. SS red blood cells havean adherent surface and attach more readily than normal cells tomonolayers of cultured tumor endothelial cells. Reticulocytes frompatients with SS disease have on their surface the integrin complex α₄β₁which binds to both fibronectin and VCAM-1, a molecule expressed on thesurface of tumor endothelial cells particularly after activation byinflammatory cytokines such as TNF, interleukins and lipid-mediatedagonists (prostacyclins). Activated tumor endothelial cells aretypically procoagulant. Similar molecules are upregulated on theneovasculature of tumors. In addition, upregulation of the adhesive andhemostatic properties of tumor endothelial cells are induced by viruses,such as herpes virus and Sendai virus. Sickled erythrocytes lackstructural malleability and aggregate in the small tortuousmicrovasculature and sinusoids of tumors. In addition, the relativehypoxemia of the interior of tumors induces aggregation of sicklederythrocytes in tumor microvasculature. Hence, sickled erythrocytes withtheir proclivity to aggregate and bind to the tumor endothelium areideal carriers of therapeutic genes to tumor cells.

The invention provides a method of delivering a therapeutic agent to asolid tumor characterized by hypoxia, acidosis and hypertonicitycomprising loading the therapeutic agent into mature sickle red bloodcells or nucleated sickle cell progenitor cell and administering thetherapeutic agent into the blood circulation of a patient wherein thesickle red blood cells accumulate in the tumor, wherein the therapeuticagent loaded into the sickle cell or sickle cell progenitor is ananti-tumor virus, toxin, siRNA, drug or prodrug.

SUMMARY OF INVENTION

The invention provides method of treating tumors using sicklederythrocytes and their nucleated precursors as carriers of therapeuticagents selectively into tumors where they induce a tumoricidal response.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided also are compositions and methods for delivery of therapeuticnucleic acid constructs to tumor sites in vivo using therapeutic genescarried by erythrocytes from patients with sickle cell anemia which havethe unique capability of adhering to sites on tumor neovasculature.

1. Cancer

This invention is used to treat any type of cancer in a host at anystage of the disease. More particularly, the cancer is a solid tumorsuch as a carcinoma, melanoma, or sarcoma. This invention is used totreat cancers of hemopoietic origin such as leukemia or lymphoma, thatinvolve solid tumors. A host is any animal that develops cancer and hasan immune system such as mammals. Thus, humans are considered hostswithin the scope of the invention.

2. Nucleic Acid

The term nucleic acid as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand.

The term isolated nucleic acid means that the nucleic acid is notimmediately contiguous with both of the sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Forexample, an isolated nucleic acid molecule can be, without limitation, arecombinant DNA molecule of any length, provided nucleic acid sequencesnormally found immediately flanking that recombinant DNA molecule in anaturally occurring genome are removed or absent. Thus, an isolatednucleic acid molecule includes, without limitation, a recombinant DNAthat exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences as well as recombinant DNA that isincorporated into a vector, an autonomously replicating plasmid, a virus(e.g., a retrovirus, adenovirus, or herpes virus), or into the genomicDNA of a prokaryote or eukaryote. In addition, an isolated nucleic acidcan include a recombinant DNA molecule that is part of a hybrid orfusion nucleic acid sequence.

Typically, regulatory elements are nucleic acid sequences that regulatethe expression of other nucleic acid sequences at the level oftranscription and/or translation. Thus, regulatory elements include,without limitation, promoters, operators, enhancers, ribosome bindingsites, transcription termination sequences (i.e., a polyadenylationsignal), and the like. In addition, regulatory elements can be, withoutlimitation, synthetic DNA, genomic DNA, intron DNA, exon DNA, andnaturally-occurring DNA as well as non-naturally-occurring DNA. It isnoted that isolated nucleic acid molecules containing a regulatoryelement are not required to be DNA even though regulatory elements aretypically DNA sequences. For example, nucleic acid molecules other thanDNA, such as RNA or RNA/DNA hybrids, that produce or contain a DNAregulatory element are considered regulatory elements. Thus, recombinantretroviruses having an RNA sequence that produces a regulatory elementupon synthesis into DNA by reverse transcriptase are isolated nucleicacid molecules containing a regulatory element even though therecombinant retrovirus does not contain any DNA.

3. Transfection

The term “transfection,” of a nucleic acid into a cell, as used hereinis intended to include “transformation,” “transduction,” “gene transfer”and the like, as they are commonly used in the art. “Transfection” isNOT intended to be limited to transfer of nucleic acid into a cell bymeans of an infectious particle such as a retrovirus, as the term mayhave been used originally. Rather any form of delivery and introductionof a nucleic acid molecule, preferably DNA, into a cell, whether in theform of a plasmid, a virus, a liposome-based vector, or any othervector, so that the nucleic acid is expressed in the cell and itsprotein product(s) made, is included within the definition of“transfection.”

When a nucleic acid is said to “encode” a product other than a protein,this language is intended to mean that it encodes the necessaryproteins/enzymes that are involved in, or required for, the synthesis ofthat product. For example, if a DNA molecule is said to encode LPS, itclearly encodes one or more proteins (enzymes) that are involved in thebiosynthesis of LPS. If a nucleic acid is said to “encode thebiosynthesis” of a structure, it means that the nucleic acid encodes theenzymes that participate in the creation of that structure. Inparticular for the carbohydrate structures referred to herein, thenucleic acids used in the invention are introduced into a cell thatnormally does not make, or makes little of, the carbohydrate structureso as to provide to that cell the genetic material for an enzyme orenzymes that generate the carbohydrate structure or modify a differentcarbohydrate structure to that one indicated.

When transfected in vitro, the cells are autologous, allogeneic, orxenogeneic to the host to provide additional immunogenicity. In additionto being transfected with nucleic acid encoding a SAg, the cells aretransfected with nucleic acid encoding any other polypeptide including,without limitation, a galactosyltransferase, staphylococcalhyaluronidase and/or erythrogenic toxin, streptococcal capsularpolysaccharide, CD44, tumor antigen, costimulatory molecule such as B7-1and B7-2, adhesion molecules, MHC class I molecule and/or MHC class IImolecule. Nucleic acids encoding the molecules are cotransfected withthe SAgs. But for others, including but not limited to Staphylococcalhyaluronidase, erythrogenic toxin, Streptococcal capsular polysaccharideand CD44 genes, the nucleic acids encoding the SAgs are fused to othernucleic acids resulting in expression of a fusion protein. Methods forin vivo and in vitro transfection of cells are well known. For example,two books in the series Methods in Molecular Medicine published byHumana Press, Totowa, N.J., describe in vivo and in vitro transfectionprotocols that are adaptable to the present invention (Vaccine Protocolsedited by Robinson et al., (1996) in Gene Therapy Protocols edited byRobbins et al., Humana Press, Totowa, N.J. (1997)). Transfectionprotocols are also discussed elsewhere ((Sambrook, J. et al., MolecularCloning, Second Edition, Cold Springs Harbor Laboratory Press,Plainview, N.Y., (1989)). In addition, use of various vectors to targetepithelial cells, use of liposomal constructs, methods of transferringnucleic acid directly into T cells, hematopoietic stem cells, andfibroblasts, methods of particle-mediated nucleic acid transfer to skincells, and methods of liposome-mediated nucleic acid transfer to tumorcells have been described elsewhere. (Felgner, P L et al., CationicLipids for Intracellular Delivery of Biologically Active Molecules, U.S.Pat. No. 5,459,127, issued Oct. 17, 1995; Felgner, P L, Cationic Lipidsfor Intracellular Delivery of Biologically Active Molecules, U.S. Pat.No. 5,264,618, issued Nov. 23, 1993; Felgner, P L, Exogenous DNASequences in a Mammal, U.S. Pat. No. 5,580,859 issued Dec. 3, 1996;Felgner, P L, A Protective Immune Response in a Mammal by Injecting aDNA Sequence, U.S. Pat. No. 5,589,466 issued Dec. 31, 1996).

Nucleic acid and nucleic acid constructs of the present invention areincorporated into a vector, an autonomously replicating plasmid, or avirus (e.g., a retrovirus, adenovirus, or herpes virus). Typically,these vectors, plasmids, and viruses can replicate and functionindependently of the cell genome or integrate into the genome. Vector,plasmid, and virus design depends on, for example, the intended use aswell as the type of cell transfected. Appropriate design of a vector,plasmid, or virus for a particular use and cell type is within the levelof skill in the art. In addition, a single vector, plasmid, or virus canbe used to express either a single polypeptide or multiple polypeptides.It follows that a vector, plasmid, or virus that is intended to expressmultiple polypeptides will contain one or more operably linkedregulatory elements capable of effecting and/or enhancing the expressionof each encoded polypeptide.

The term “operably linked” means that two nucleic acid sequences are ina functional relationship with one another. For example, a promoter (orenhancer) is operably linked to a coding sequence if it effects (orenhances) the transcription of the coding sequence. A ribosome bindingsite is operably linked to a coding sequence if it is positioned tofacilitate translation. Operably linked nucleic acid sequences are oftencontiguous, but this is not a requirement. For example, enhancers neednot be contiguous with a coding sequence to enhance transcription of thecoding sequence.

A vector, plasmid, or virus that directs the expression of a polypeptidesuch as a SAg can include other nucleic acid sequences such as, forexample, nucleic acid sequences that encode a signal sequence or anamplifiable gene. Signal sequences are well known in the art and can beselected and operatively linked to a polypeptide encoding sequence suchthat the signal sequence directs the secretion of the polypeptide from acell. An amplifiable gene (e.g., the dihydrofolate reductase [DHFR]gene) in an expression vector can allow for selection of host cellscontaining multiple copies of the transfected nucleic acid.

Standard molecular biology techniques are used to construct, propagate,and express the nucleic acid, nucleic acid constructs, vectors,plasmids, and viruses of the invention ((Sambrook, J. et al., supra;Maniatis et al., Molecular Cloning (1988); and U.S. Pat. No. 5,364,934.For example, prokaryotic cells (e.g., E. coli, Bacillus, Pseudomonas,and other bacteria), yeast, fungal cells, insect cells, plant cells,phage, and higher eukaryotic cells such as Chinese hamster ovary cells,COS cells, and other mammalian cells can be used.

4. Sickled Erythrocytes as Gene Carriers

Erythrocytes from patients with sickle cell anemia contain a highpercentage of SS hemoglobin which under conditions of deoxygenationaggregate followed by the growth and alignment of fibers transformingthe cell into a classic sickle shape. Retardation of the transit time ofsickled erythrocytes results in vaso-occlusion. SS red blood cells havean adherent surface and attach more readily than normal cells tomonolayers of cultured tumor endothelial cells. Reticulocytes frompatients with SS disease have on their surface the integrin complex α₄β₁which binds to both fibronectin and VCAM-1, a molecule expressed on thesurface of tumor endothelial cells particularly after activation byinflammatory cytokines such as TNF, interleukins and lipid-mediatedagonists (prostacyclins). Activated tumor endothelial cells aretypically procoagulant. Similar molecules are upregulated on theneovasculature of tumors. In addition, upregulation of the adhesive andhemostatic properties of tumor endothelial cells are induced by viruses,such as herpes virus and Sendai virus. Sickled erythrocytes lackstructural malleability and aggregate in the small tortuousmicrovasculature and sinusoids of tumors. In addition, the relativehypoxemia of the interior of tumors induces aggregation of sicklederythrocytes in tumor microvasculature. Hence, sickled erythrocytes withtheir proclivity to aggregate and bind to the tumor endothelium areideal carriers of therapeutic genes to tumor cells.

Red blood cell mediated transfection is used to introduce variousnucleic acids into the sickled erythrocytes. The extremely plasticstructure of the erythrocyte and the ability to remove its cytoplasmiccontents and reseal the plasma membranes enable the entrapment ofdifferent macromolecules within the so-called hemoglobin free “ghost.”Combining these ghosts and a fusogen such as polyethylene glycol haspermitted the introduction of a variety of macromolecules into mammaliancells (Wiberg, F C et al., Nucleic Acid Res. 11: 7287-7289 (1983);Wiberg, F C et al., Mol. Cell. Biol. 6: 653-658 (1986); Wiberg, F C etal., Exp. Cell. Res. 173: 218-227 (1987). Both transient and stableexpression of introduced DNA is achieved by this method. Sickled cellscan also be transfected with a nucleic acid of choice e.g.,apolipoproteins, RGD in the nucleated prereticulocyte phase (e.g.proerythroblast or normoblast stage) by methods given in Example 1.Sickled erythrocytes transfected with nucleic acids encoding a SAgand/or carbohydrate modifying enzyme to induce expression of the a Galepitope, apolipoproteins, RGD and/or any construct described herein.Nucleic acids encoding additional polypeptides alone or together withSAg as described in Tables I and II to including but not limited toangiostatin, apolipoproteins, RGD, streptococcal or staphylococcalhyaluronidase, chemokines, chemoattractants and Staphylococcal protein Aare transfected into and expressed by sickled erythrocytes. Thesesickled cell transfectants are administered parenterally and localize totumor neovascular endothelial sites where they induce a anti-tumorresponse. Protocols for use of these transfectants in the induction ofanti-tumor immune response are described in Examples 3, 4, 5, 6, 7.

5. Vesicles from Sickled Erythrocytes

Vesicles from sickled erythrocytes are shed from the parent cells. Thecontain membrane phospholipids which are similar to the parent cells butare depleted of spectrin. They also demonstrate that a shortenedRussell's viper venom clotting time by 55% to 70% of control values andbecome more rigid under acid pH conditions. Rigid sickle cell vesiclesinduce hypercoagulability, are unable to pass through the spleniccirculation from which they are rapidly removed. Sickled erythrocytesare transfected in the nucleated prereticulocyte phase with superantigenand apolipoprotein nucleic acids as well as RGD nucleic acids. Nucleicacids encoding additional polypeptides alone or together with SAg asdescribed in Tables I and II are transfected into and expressed bysickled erythrocytes. Any of the immature or mature sickled erythrocytesand their shed vesicles expressing the molecules given in Tables I andII are capable of localizing to tumor microvascular sites where theybind to apolipoprotein receptors and induce an anti-tumor effect.Because of their adhesive and hypercoagulable properties as well astheir rigid structure, these sickled cell vesicles expressingsuperantigen and apolipoproteins are especially useful for targeting thetumor microvascular endothelium and producing a prothrombotic,inflammatory anti tumor effect. Sickled erythrocytes and their vesiclesare capable of acquiring oxyLDL via fusion with oxyLDL containingliposomes as in Example 5. The resulting sickle cell or liposomeexpresses oxyLDL alone or together with SAg. Binding of oxyLDL to theSREC receptor on tumor microvascular endothelial cells induces apoptosisand simultaneous superantigen deposition produces a potent T cellanti-tumor effect.

Vesicles are prepared and isolated as follows: Blood is obtained frompatients with homozygous sickle cell anaemia. The PCV range is 20-30%,reticulocyte range is 8-27%, fetal hemoglobin range is 25-13% andendogenous level of ISCs is 2-8%. Blood is collected in heparin and thered cells are separated by centrifugation and washed three times with09% saline. Cells are incubated at 37° C. and 10% PCV in Krebs-Ringersolutions in which the normal bicarbonate buffer is replaced by 20 mMHepes-NaOH buffer and which contains either 1 mM CaCl₂ or 1 mM EGTA. Allsolutions contain penicillin (200 u/mI) and streptomycin sulphate (100ug/ml). Control samples of normal erythrocytes are incubated in parallelwith the sickle cells. Incubations of 10 ml aliquots are conducted ineither 100% N₂ or in room air for various periods in a shaking waterbath (100 oscillations per mm). N₂ overlaying is obtained by allowingspecimens to equilibrate for 45 mm in a sealed glove box (Gallenkamp)which was flushed with 100% N₂. Residual oxygen tension in the sealedbox was less than 1 mmHg. The percentage of irreversibly sickled cellsis determined by counting. 1000 cells after oxygenation in room air for30 mm and fixation in buffered saline (130 mM Cl, 20 mM sodiumphosphate, pH 74) containing 2% glutaraldehyde. Cells whose length isgreater than twice the width and which possessed one or more pointedextremities under oxygenated conditions are considered to beirreversibly sickled. After various periods of incubation, cells aresedimented at 500 g for 5 mm and microvesicles) are isolated from thesupernatant solution by centrifugation at 15,000 g for 15 mm. Themicrovesicles form a firm bright red pellet sometimes overlain by apink, flocculent pellet of ghosts (in those cases where lysis wasevident) which is removed by aspiration. Quantitation of microvesiclesis achieved by resuspension of the red pellet in 1 ml of 05% Triton X100followed by measurement of the optical density of the clear solution at550 nm. Optical density measurements at 550 nm give results that arerelatively the same as measurements of phospholipid and cholesterolcontent in the microvesicles. Cell lysis is determined by measurement ofthe optical density at 550 nm of the clear supernatant solutionremaining after sedimentation of the microvesicles. Larger samples ofmicrovesicles for biochemical and morphological analysis are preparedfrom both sickle and normal cells following incubation of up to 100 mlof cell suspension at 37° C. for 24 h in the absence or presence of Ca₂Ghosts are prepared from sickle cells after various periods ofincubation. The cells are lysed and the ghosts washed in 10 mM Tris HClbuffer, pH 73, containing 0.2 mM EGTA.

These vesicles are useful as a preventative or therapeutic vaccine as inExamples 4, 5, 6, 7.

6. Sickled Erythrocytes as Carriers of Tumoricidal Agents.

Sickled erythrocytes are known to be more adherent to microvascularendothelium than normal erythrocytes and to adhere to a greater extentunder conditions of local hypoxia and acidosis. The primary pathologicdefect in sickle cell disease is the abnormal tendency of hemoglobin Sto polymerize under hypoxic conditions. The polymerization ofdeoxygenated hemoglobin S results in a distortion of the shape of thered cell and marked decrease in its deformability. These rigid cells areresponsible for the vaso-occlusive phenomena which are the hallmark ofthe disease.

Sickle red cells adhere to the microvascular endothelium for thefollowing reasons: Sickled cells have abnormally increased expression ofα₄β₁ integrin and CD36. Activation of platelets releases thrombospondin,which act as a bridging molecule by binding to a surface molecule, CD36,on an endothelial cell and to CD36 or sulfated glycans on a sicklereticulocyte. Inflammatory cytokines induce the expression ofvascular-cell adhesion molecule 1 (VCAM-1) on endothelial cells. Thisadhesive molecule binds directly to the α₄β₁ integrin on the sicklereticulocyte.

In the oxygenated state, the extent of sickle cell adhesion isdensity-class dependent: reticulocytes and young discocytes (SS1)greater than discocytes (SS2) greater than irreversible sickle cells andunsicklable dense discocytes (SS4). Hypoxemic conditions have no effecton adherence of normal erythrocytes but sickle erythrocyte adherence toendothelial cells is increased significantly. The least dense sickleerythrocytes containing CD36 and VLA-4+ expressing reticulocytes areespecially involved in hypoxia sensitive adherence. Selective secondarytrapping of SS4 (dense cells) occurs in post capillary venules wheredeformable SS cells are preferentially adherent. Vaso-occlusion isinduced by a combination of precapillary obstruction, adhesion in postcapillary venules, and secondary trapping of dense erythrocytes. Thisinduces local hypoxia leading to increased polymerization of hemoglobinS and rigidity of SS erythrocytes. In this way the obstruction ismultiplied and extended to nearby vessels.

In the present invention, sickled erythrocytes are used to carrytumoricidal agents into the microvasculature of tumors. Sickle celltrait cells are preferred since they are normal under physiologicconditions but sickle and become adhesive in the acidotic and/orhypoxemic tumor microvasculature. Tumoricidal agents introduced into andcarried by sickled erythrocytes include oncolytic viruses including butnot limited to herpes simplex, adenoviruses, vaccinia, Newcastle Diseasevirus, autonomous parvoviruses, In addition, the adenovirus encodingthymidine kinase is transfected into tumor cells that are thensusceptible to lysis ganciclovir. Various oncolytic and tumor specificviruses with tumor specificity used to transfect sickle cells aredescribed in Table 1 of Kirn, D. et al., Nat. Med. 7:781-7 (2001) shownbelow.

TABLE 1 Examples of replication-selective viruses in clinical trials forcancer patients Clinical Tumor targets Cell phenotype allowing ParentalStrain Agent phase in clinical trials Genetic alterations selectivereplication Engineered Adenovirus d/1520^(a) I-III SCCHN E1B-55-kD genedeletion Controversial cells lacking p53 function (2/5 chimera)Colorectal (for example, deletion, mutation), other? Ovarian PancreaticE3-10.4/14.5 deletion Adenovirus CN706 I E1A expression driven by PSEelement Prostate cells (malignant, normal) (serotype S) CN787 I ProstateE1A driven by rat probasin promoter/ E1B by PSE/promoter/enhancerAdenovirus Ad5-CD/tk-rep I Prostate E1B-55-kD gene deletionControversial cells lacking p53 function (2/5 chimera) Insertion ofHSV-tk/CD fusion gene (for example, deletion, mutation), other? Herpessimplex G207 I-II GBM ribonucleotide reductase disruption Proliferatingcells virus-1 (lacZ insertion into ICP6 gene) neuropathogenesis genemutation (γ-34.5 gene)-both copies Herpes simplex NV1020 I Colorectalneuropathogenesis gene mutation Proliferating cells virus-1 (γ-34.5gene)-single copy Vaccinia virus Wild-type ± I Melanoma For selectivity:none or tk deletion Unknown GM-CSF Immunostimulatory gene (GM-CSF)Insertion Non-engineered Newcastle 73-T I Bladder Unknown Loss of IFNresponse in tumor cells Disease virus SCCHN (serial passage on tumorcells) Ovarian Autonomous H-1 I None Transformed cells parvoviruses ↑proliferation ↓ differentiation ras, p53 mutation Reovirus Reolysin ISCCHN None Ras-pathway activation (for example, ras mutation, EGFRsignaling)

Erythrocytes from subjects with sickle trait are preferred because thesered cells are functionally and structurally normal in the circulationbut are activated to sickle in the hypoxic tumor vasculature. Here theyassume the sickled configuration, adhere to the endothelium of the tumormicrocirculation and obstruct microvasculature in a manner similar tothe homozygous SS erythrocytes.

In addition the sickled erythrocyte carry nucleic acids encodingtumoricidal agents including but not limited to C. perfringens exotoxin,pertussis toxin, verotoxins, pseudomonas exotoxins and superantigens,perforin, granzyme B, complement components (membrane attack complex),oxidized LDL, tumor specific antibodies alone or fused to toxinsincluding but not limited to superantigens, Pseudomonas exotoxins,ricin, clostridia toxin. The nucleic acid encodes a hemolysin such asbut not limited to E. coli hemolysin or staphylococcal alpha hemolysin.The sickled cell can also contain anaerobic bacterial spores such asclostridia species which can grow selectively in hypoxemic tissues. Thesickled erythrocyte also carries phage displays, exosomes, sickle cellvesicles, sec vesicles expressing tumor toxins or superantigens. Thetoxins may be fusion proteins of toxins with ligands expressed on tumorvasculature or tumor such a EGF, inactivated factor VIII or antibodiesspecific for a wide variety of tumor antigens well known in the art.

The nucleic acids encoding these toxins and oncolytic and tumor specificviruses are placed under the promoter of the heat sensitive globaloperator (Example 8). When entering the hypoxic tumor, sicklederythrocyte adhere to the tumor vasculature. In the hypoxemicenvironment of the tumor, the hypoxia sensitive global promoter isactivated and induces the production lytic viruses and toxins. Sickledcells are disrupted and lyse releasing lytic virus and toxin into thehypoxic tumor. As the tumor site becomes more hypoxic, VCAM-1 andp-selectin expression on tumor endothelium are upregulated trapping morecirculating sickled cells in the tumor microcirculation to undergo lysiswith release of tumoricidal products into the tumor area.

The sickled cell is transfected preferably with the oncolytic virusesand toxins given above at a stage preferably before it is enucleated(Examples 1, 8). Nucleated sickle reticulocytes are the preferred cellfor transfection although enucleated sickled cells will also work(Example 8) Anaerobic bacterial spores such clostridia are transfectedinto the sickled erythrocytes by endocytosis or electroporation (SchrierS. Methods in Enzymology 149: 261-271 (1987); Tsong T Y Methods inEnzymology 149-259 (1987)). They are also introduced into sickleerythrocytes that have been lysed under hypotonic conditions and themembranes annealed with encapsulation of the anaerobic spores (Example8).

Erythrocytes from subjects with sickle trait are preferred because thesered cells are functionally and structurally normal in the circulationbut are activated to sickle in the hypoxic tumor vasculature. Here theyassume the sickled configuration, adhere to the endothelium of the tumormicrocirculation and obstruct microvasculature in a manner similar tothe homozygous SS erythrocytes.

The sickled erythrocytes are administered parenterally by injection orinfusion in a therapeutically effective amount of cells. Thisencompasses a volume of 1-25 cc of packed cells administered i.v. over aone hour period. These cells are used in protocols given in Example 3-7.

Another preferred delivery system is the sickled erythrocyte containingthe nucleic acids of choice a given in Example 6. The sicklederythrocytes undergo ABO and RH phenotyping to select compatible cellsfor delivery. The cells are delivered intravenously or intrarterially ina blood vessel perfusing a specific tumor site or organ e.g. carotidartery, portal vein, femoral artery etc. over the same amount of timerequired for the infusion of a conventional blood transfusion. Thequantity of cells to be administered in any one treatment would rangefrom one tenth to one half of a full unit of blood. The treatments aregenerally given every three days for a total of twelve treatments.However, the treatment schedule is flexible and may be given for alonger of shorter duration depending upon the patients response.

TABLE I Therapeutic Constructs And Preferred Conditions Of Use I. CELLS:Tumor Cells, DCs or DC/Tumor Cell Hybrids (DC/tc) USE: In vivo and Exvivo PURPOSE A. In Vivo Preventative or Therapeutic Vaccine (EstablishedTumor) Accomplish by transfecting or co-transfecting with nucleic acidencoding superantigen plus one or more of the following: 1.Superantigens 2. Enzyme that modifies carbohydrate to induce Gal orGalCer epitope expression 3. Functional hyaluronidase from microbial orhuman sources 4. Staphylococcal or streptococcal erythrogenic toxin 5.Staphylococcal protein a or a domain thereof 6. Staphylococcal hemolysinand functional microbial toxins 7. Functional microbial or humancoagulase 8. Costimulatory protein 9. Chemoattractants 10. Chemokines11. Nucleic acids encoding biosynthesis of lipopolysaccharides 12.Nucleic acids encoding biosynthesis of glycosylceramides 13. Nucleicacids encoding biosynthesis of microbial membrane or capsularlipoproteins and polysaccharides 14. Oncogenes, amplified oncogenes andtranscription factors 15. Angiogenic factors and receptors 16. Tumorgrowth factor receptors 17. Tumor suppressor receptors 18. Cell cycleproteins 19. Heat-shock proteins, ATPases and G proteins 20. Proteinsengaged in antigen processing, sorting and intracellular trafficking 21.Inducible nitric oxide synthase (iNOS) 22. apolipoproteins (e,g,. Lp(a))transfected into tumor cells & sickled erythrocytes used for targetingtumor microvasculature 23. LDL and oxyLDL receptors (e.g., SCEPreceptor) transfected into tumor cells and sickled erythrocytes & usedfor targeting to tumor microvasculature B. Ex Vivo Immunization of Tand/or NKT cells to Produce Tumor Specific Effector Cells (for AdoptiveImmunotherapy)* Accomplish by (i) transfecting or co-transfecting tumoror accessory cells with nucleic acid encoding the following, or (ii)providing immobilized molecules or receptors that present thefollowing: 1. Superantigen 2. Superantigen receptor and transcriptionfactor with bound superantigen 3. CD1 receptor binding and/or expressingsuperantigen-glycosyl ceramide complex 4. CD14 receptor binding orexpressing superantigen-lipopolysaccharide or superantigen-peptidoglycan complex 5. Mannose receptor binding glycosylatedsuperantigen 6. Glycophorin receptor 7. Superantigen-tumor peptide(s)complex on MHC or CD1-bearing APC in soluble or immobilized form C.Therapeutic Molecules or Complex Applied to Transfected or UntransfectedTumor cells or Accessory Cells; or MHC class I, class II, CD1,Superantigen receptor or CD14 receptor: 1. Superantigen (wherein cellmay express Gal) 2. Glycosylated superantigen 3. Superantigen complexwith a. glycosyl ceramide b. lipopolysaccharide c. peptidoglycan d.mannan proteoglycan e. muramic acid f. tumor peptide g.glycosylceramides with terminal Gal(α1-4)Gal e.g. globotriosylceramideand galabiosylceramide h. Conjugates of SAg-(Gb2 or Gb3 or Gb4) i.Conjugates of SAg-(Gb2 or Gb3 or Gb4)-CD1 j. GPI anchored conjugates:SAg-GPI-(Gb2 or Gb3 or Gb4) l. GPI anchored conjugates: SAg-GPI-(Gb2 orGb3 or Gb4)-CD1 m. Conjugates of SAg polypeptide or nucleic acid withVerotoxin n. Conjugates of SAg Polypeptide or nucleic acid withVerotoxin A or B subunit o. Conjugates of SAg polypeptide or nucleicacid with IFNα receptor peptides homologous to verotoxin p. Conjugatesof SAg polypeptide or nucleic acid with CD19 peptides homologous toverotoxin q. Conjugates of SAg polypeptide or nucleic acid withArg-Gly-Asp or Asn-Gly-Arg r. Conjugates of SAg polypeptide or nucleicacid with LDL, VLDL, HDL s. Conjugates of SAg polypeptide or nucleicacid with Apolipoproteins (e.g., Lp(a), apoB- 100, apoB-48, apoE) t.Conjugates of SAg polypeptide or nucleic acid with oxyLDL, oxyLDLmimics, (e.g., 7β- hydroperoxycholesterol, 7β-hydroxycholesterol,7-ketocholesterol, 5α-6α- epoxycholesterol,7β-hydroperoxy-choles-5-en-3β-ol, 4-hydroxynonenal (4-HNE), 9- HODE,13-HODE and cholesterol-9-HODE) u. Conjugates of SAg polypeptide ornucleic acid with oxyLDL by products (e.g. lysolecithin,lysophosphatidylcholine, malondialdehyde, 4-hydroxynonenal) v. LDL &oxyLDL receptors (e.g., LDL oxyLDL, acetyl-LDL, VLDL, LRP, CD36, SREC,LOX-1, macrophage scavenger receptors) as polypeptide or nucleic acidalone or with SAg polypeptide or nucleic acid intratumorally II. CELLS:Specialized Tumor Specific Effector Cells (T and/or NKT Cells) USE:Adoptive Immunotherapy In Vivo PURPOSE: A. CD44 Expression on T cells orNKT Accomplished by: (i) Superantigen stimulation; and/or (ii)transfection with nucleic acid encoding CD44 and/or (iii) transfectionwith nucleic acid encoding glycosyltransferase B. Chimeric TCR with:Invariant a chain site for binding GalCer and Vβ chain site for bindingsuperantigen C. Dual TCR Vβ chains with sites for superantigen bindingD. T cells or NKT cells with overexpressed Vβ region specific for agiven superantigen E. T cells or NKT cells with lowered signaltransduction threshold III. MOLECULES: Superantigen mimics USE: In VivoAdministration A. Superantigen receptor-binding oligonucleotides B.Superantigen oligonucleotide-peptide conjugate Oligonucleotide isspecific for superantigen receptor on tumor cells Peptide has deletedclass II binding site and intact TCR binding site C. Phage displayedintegrin ligand on tumor neovasculature - carrier forsuperantigen-encoding nucleic acid. IV. CARRIERS: for nucleic acidencoding superantigen USE Transfection of Tumors In vivo A. Sicklederythrocytes that target tumor neovasculature B. Phage displayed tumorneovascular integrin and superantigen receptor carrying superantigennucleic acids V. CARRIERS: constructed to co-express superantigenconjugates or complexes with: Glycosylceramide αGal LipopolysaccharidesPeptidoglycans USE Transfection of Tumor Cells and/or DCs and/orDC/tc's - in vivo or ex vivo. A. Liposomes B. Proteosomes

TABLE II Nucleic Acid Constructs and Cells SAg-encoding DNA is usedalone or together with DNA encoding other cell surface moieties usefulin generating antitumor immunity. Genes or their products are shown incolumn 1, source information is shown in column 3, preferred cells to betransformed, transfected or transduced with the DNA are shown in column2. All of references are incorporated by reference in their entirety.Gene or Gene Product Cells transformed Reference or Source 1. SAg (SEQID NOS: 1-2) Tumor [See text] 2. Enterotoxin (SEQ ID NOS (3-12) Tumor[See text] 3. SAg receptor (SEQ ID NOS 1-2) Tumor [See text] 4.Enterotoxin receptor Tumor [See text] (SEQ ID NOS 3-12) 5. CD1receptor(s) (SEQ ID NO 13-14) Tumor Martin, L H et al., Proc. Natl.Acad. Sci. 83: 9154-9158 (1986) 6. CD14 receptor (SEQ ID NOS 15-16)Tumor Ferrero, E et al., J. Immunol. 145: 331-336 (1990) 7. CD44encoding nucleic acids T or NKT Nottenburg, C et al. Proc. (SEQ ID NO17) Natl. Acad. Sci. 66: 8521-88525 (1992) 8. Carbohydrate modifyingenzymes Tumor, T or NKT Sheng, Y et al. Int. J. Cancer (SEQ ID: NO 18)73: 850-858 (1997) 9. TCR Vβ chain (SEQ NOS 19-20) Tumor Tillinghast, JP et al., Science 233: 879-883 (1986) 10. Staph/Strep hyaluronidaseTumor Hynes W L et al., Infect. (SEQ NOS: 21-22) Immun., 63: 3015-3020(1995) 11. Staph/Strep erythrogenic toxin Tumor McShan W M, et al., Adv.(SEQ NOS 23-24) Exp. Med. Biol. 418: 971-973 (1997) 12. Staphylococcalβ-hemolysin Tumor Projan S J et al., Nucleic Acid (SEQ NOS: 25-26) Res.3305-3309 (1989) 13. Strep capsular polysaccharide Tumor Lin, W S etal., J. (SEQ NOS: 27-28) Bacteriol. 176: 7005-7016 (1994) 14. Staphstaphylocoagulase Tumor Kaida S. et al., J. (SEQ NOS 29-30) Biochemistry102: 1177-1186 (1987) 15. Staph Protein A (SEQ NOS: 31-32) TumorShuttleworth, H L et al., Gene 58: 283-295 (1987) 16. Staph Protein Adomain D Tumor Roben, P W et al., J. (SEQ NOS: 33-34) Immunol. 154:6347-6445 (1995) 17. Staph Protein A Domain B Tumor Gouda, H et al.,(SEQ NO: 35) Biochemistry, 31: 9665-9672 (1992) 18. Immunostimulatoryprotein Tumor, T or NKT Tokunaga, T et al., Microbiol. Immunol. 36:55-66, (1992) 19. Costimulatory protein Tumor Entage, P C et al., J.Immunol. 160: 2531-2538 (1998) 20. SAg-mimicking nucleic acid T or NKT21. Glycophorin (SEQ NOS: 36-37) Tumor Siebert, P D. et al., Proc. Natl.Acad. Sci. USA 83 1665-1669 (1986) 22. Mannose receptor Tumor Kim S J.et al., Genomics 14: (SEQ ID NOS 38-39) 721-727 (1992) 23. Angiostatin(SEQ ID NO: 40) Tumor Cao, Y. et al., J. Clin. Invest 101: 1055-1063(1998) 24. Chemoattractant Tumor Ames, R S. et al., J. Biol. (SEQ IDNOS: 41-42) Chem. 271: 20231-20234 (1996) 25. Chemokine (SEQ ID NOS43-44) Tumor Nagira, M et al., J. Biol. Chem. 272: 19518-19524 (1997)26. Transcription factor (SEQ ID NO 45) Tumor, T or NKT Schwab M et al.,Mol. Cell Biol. 6: 2752-2758 (1986) 27. Transcription factor-bindingTumor, T or NKT nucleic acid 28. SAg/peptide conjugate Tumor 29.Glyco-SAg Tumor 30. Staph. global regulator gene agr Tumor Balaban, N.et al., Proc. Natl. (SEQ ID NO: 46-48) Acad. Sci. USA 92: 1619-1623(1995) 31. Lipid A biosynthetic genes Tumor Schnaitman C A et al., gelpxA-D (SEQ ID NOS: 49-56) Microbiological Reviews 57: 655-682 (1993)32. Mycobacterial mycolic acid Tumor Fernandes N D et al., Genebiosynthetic genes 170: 95-99 (1996); Mathur M (SEQ ID NOS: 57-58) etal., J. Biol. Chem. 267: 19388-19395 (1992) 33. c-abl oncogene amplifiedin Tumor Scherle P A et al., chronic myel. Leukemia Proc. Natl. Acad.Sci. USA (SEQ ID NOS: 59-60) 87: 1908 (1990); Heisterkamp N et. al.,Nature 344: 251-253 (1990) 34. erbB2 (HER2/neu) oncogene Tumor SchechterA L et al., Science (SEQ ID NOS: 61-62) 229: 976 (1985); Bargmann C LNature 319: 226 (1986); Hung M C et al., Proc. Natl. Acad Sci. 83: 261(1986); Yamamoto T et al., Nature 319: 230 (1986) 35. IGF-1 receptorgene Tumor Abbott A M et al., J. Biol. (SEQ ID NOS: 63-64) Chem. 267:10759-10763 (1992); Scott J et al., Nature 317: 260-262 (1985); Liu J etal., Cell 75: 59-63 (1993) 36. VEGF Tumor Tischer E et al., J. Biol.(SEQ ID NOS: 65-66) Chem. 266: 11947-11954 (1991) 37. Strep emm-likegene family Tumor Kehoe M A, In: Cell-Wall Associated Proteins inGram-Positive Bacteria in Bacterial Cell Wall, Ghuysen J M et al., eds,Elsevier, Amsterdam, 1994 38. iNOS (SEQ ID NOS 67-68) Tumor Xie Q W etal., Science 256: | 225-228 (1992) 39. Apolipoproteins (e.g., Lp(a),Tumor [See Text] apoB-100, apoB-48, apoE) (SEQ ID NOS: 69-74) 40. LDL &oxyLDL receptors Tumor [See Text] (e.g., LDL oxyLDL, acetyl-LDL, VLDL,LRP, CD36, SREC, LOX-1, macrophage scavenger receptors) (SEQ ID NOS:75-86)6. Superantigens (SAgs)

SAgs are polypeptides that have the ability to stimulate large subsetsof T cells. SAgs include Staphylococcal enterotoxins, Streptococcalpyrogenic exotoxins, Mycoplasma antigens, rabies antigens, mycobacteriaantigens, EB viral antigens, minor lymphocyte stimulating antigen,mammary tumor virus antigen, heat shock proteins, stress peptides, andthe like. Any SAg can be used as described herein, although,Staphylococcal enterotoxins such as SEA, SEB, SEC, and SED andstreptococcal pyrogenic exotoxins such as toxic shock-associated toxin(TSST-1 also called SEF) are preferred.

When using enterotoxins, the region related to emetic activity can beomitted to minimize toxicity. In addition, SAgs can be derivatized tominimize toxicity. The level of toxicity may not be a concern when usingSAg transfected cells to activate lymphocytes ex vivo since thelymphocytes can be rinsed of SAg polypeptide prior to administration toa host.

The nucleic acid sequences that encode SAgs are known and readilyavailable. For example, Staphylococcal enterotoxin A (SEA), SEB, SEC,SED, SEE, TSST-1, and Streptococcal pyrogenic exotoxin (SPEA) have beencloned and can be expressed in E. coli (Betley M J and J J Mekalonos, J.Bacteriol. 170:34 (1987); Huang I Y et al., J. Biol. Chem., 262:7006(1987); Betley M et al., Proc. Natl. Acad. Sci. USA, 81:5179 (1984);Gaskill M E and SA Khan, J. Biol. Chem., 263:6276 (1988); Jones C L andSA Khan, J. Bacteriol., 166:29 (1986); Huang I Y and MS Bergdoll, J.Biol. Chem., 245:3518 (1970); Ranelli D M et al., Proc. Nat. Acad. Sci.USA 82:5850 (1985); Bohach G A, Infect Immun., 55:428 (1987); Bohach GA, Mol. Gen. Genet. 209:15 (1987); Couch J L et al., J. Bacteriol.170:2954 (1988); Kreiswierth B N et al., Nature, 305:709 (1983); CooneyJ et al., J. Gen. Microbiol., 134:2179 (1988); Iandolo J J, Annu. Rev.Microbiol., 43:375 (1989); and U.S. Pat. No. 5,705,151)). Additionalnucleic acid sequences encoding SAgs are described elsewhere (Bohach etal., Crit. Rev. in Microbiology 17:251-272 (1990); (Kotzin, B L et al.,Advances Immunology 54: 99-165 (1993))

PCR can be used to isolate SAg-encoding acid. For example, the nucleicacid encoding SEA, SEB, and TSST-1 can be isolated as describedelsewhere (Dow et al., J. Clin. Invest. 99:2616-2624 (1997)). Briefly,the following primers can be used to amplify the SAg-encoding nucleicacid:

SEA forward: GGGAATTCCATGGAGAGTCAACCAG, (SEQ ID NO: 87) SEA backward:GCAAGCTTAACTTGTTAATAG; (SEQ ID NO: 88) SEB forward:GGGAATTCCATGG-AGAAAAGCG, (SEQ ID NO: 89) SEB backward:GCGGATCCTCACTTTTTCTTTG; (SEQ ID NO: 90) TSST-1 forward:GGGGTACCCCGAAGGAGGAAAAAAAAA (SEQ ID NO: 91) TGTCTACAAACGATAATATAAAG,TSST-1 backward: TGCTCTAGAGCATTAATTAATTTCTGC (SEQ ID NO: 92)TTCTATAGTTTTTAT

The full-length TSST-1 nucleic acid sequence is cloned into a eukaryoticexpression vector (pCR3; InVitrogen Corp., San Diego, Calif.), whereasonly the sequence corresponding to the mature SEB and SEA (sequencesminus the putative bacterial signal sequences) is cloned into pCR3.Removal of the SEB and SEA signal sequences increases the level ofexpression in transfected cells. The plasmids are grown in Escherichiacoli and plasmid DNA extracted by the modified alkaline lysis method andpurified on a CsCl gradient.

Nucleic acids encoding mutant or variant SAgs are also considerednucleic acid sequences encoding SAgs within the scope of the invention.For example, a mutant SAg-encoding acid sequence is engineered such thatthe resulting SAg is devoid of amino acid residues, e.g., histidine,known to produce toxicity Likewise, SAg-encoding nucleic acid isengineered to contain or lack sequences that facilitate the selectivebinding of SAgs to certain Vβ regions of the TCR present on T cells orto ganglioside, mannose (or other carbohydrate) receptor, certainregions of MHC class II, and/or enterotoxin receptors present on tumorcells, antigen presenting cells (APCs), and/or lymphocytes.

Nucleic acid sequences that encode a SAg are also fused, in frame, withnucleic acid that encodes another polypeptide. This larger nucleic acidis termed herein a SAg fusion gene and the resulting polypeptide productis a SAg fusion product. Nucleic acid sequences that are fused toSAg-encoding nucleic acid include, without limitation, nucleic acidsequences that encode tumor antigens, costimulatory molecules, adhesionmolecules and MHC class II molecules. The superantigen fusion product issecreted by a transfected cell, expressed on the cell surface or it mayremain intracellular in nucleic acid or partly processed form.

SAgs are also isolated and purified from their natural source as well asfrom a heterologous expression system such as E. coli. Likewise,SAg-containing polypeptides (e.g., SAg fusion products) are isolated andpurified from a heterologous expression system. In addition,Staphylococcus strains producing high levels of enterotoxin have beenidentified and are available. For example, exposingenterotoxin-producing Staphylococcus aureus to mutagenic agents such asN-methyl-N-nitro-N-nitrosoguanidine results in a 20 fold increase inenterotoxin production over the amounts produced by the parent wild-typeStaphylococcus aureus strain (Freedman M A and Howard M B J. Bacteriol.,106:289 (1971)).

7. Tumor Cells or Sickled Erythrocytes and Vesicles Expressing SAg andApolipoproteins

Superantigen nucleic acids are fused in frame to nucleic acids encodingapoproteins including but not limited to apoproteins Lp(a), B-48 and 100and E3 and transfected into tumor cells in vivo to produce tumor cellsexpressing superantigens and apoproteins. These tumor cells arerecognized by apoprotein receptors in tumor microvasculature. Tumorcells are also transfected ex vivo with the identical nucleic acidconstructs. A RGD sequence is added to promote deposition in the tumormicrovasculature which are useful. These tumor cell transfectantsexpressing Sag, apoprotein and RGD bind to apoprotein receptors andintegrins respectively expressed in tumor microvasculature wherein theyinitiate a potent and localized anti-tumor response.

Superantigen nucleic acids together with nucleic acids encoding eitherapo(a), apoB and apoE4 are also transfected into nucleated sicklederythrocytes (e.g., proerythroblast or normoblast phase) by methodsgiven in Examples 1 and 6. The integrin ligand RGD nucleic acids aretransfected into tumor cells or sickled cells to facilitate thelocalization of the transfected tumor cells and sickled cells tointegrins expressed in the tumor neovasculature in vivo (see Example 6).Alternatively, the sickled erythrocytes or tumor cells acquire theapolipoprotein or oxyLDL by coculture with liposomes which express theapolipoprotein or oxyLDL (see Section 7 & Example 5).

These tumor cells or sickle cell transfectants are administeredparenterally and are capable of trafficking to tumor microvasculaturewherein they bind to apolipoprotein and scavenger receptors onendothelial cells and macrophages. The transfectants are phagocytosed bymacrophages cells and induce endothelial cell apoptosis. SAgs expressedon the tumor cells and sickle cells also induce a local T cellinflammatory anti-tumor response which envelops the neighboring tumorcells.

These tumor cell and sickle cell constructs are prepared by methodsgiven in Examples 1 and 6 and are useful in vivo against primary and/ormetastatic tumors according to Examples 3-7.

8. Functional Homologues & Derivatives of Proteins of Peptides

All of the protein and nucleic acid compositions given herein areintended to encompass functional derivatives. All of the functionalderivatives of the fusion partners for superantigens described in thisapplication are encompassed by this invention. Similarly, Staphylococcalenterotoxins or superantigens are intended to encompass functionalderivatives of a particular superantigen or enterotoxin.

By “functional derivative” is meant a “fragment,” “variant,”“homologue,” “analogue,” “fusion protein,” or “chemical derivative”,which terms are defined below. A functional derivative retains at leasta portion of the function of the native protein monomer which permitsits utility in accordance with the present invention.

A “fragment” refers to any shorter peptide. A “variant” of refers to amolecule substantially similar to either the entire protein or a peptidefragment thereof. Variant peptides may be conveniently prepared bydirect chemical synthesis of the variant peptide, using methodswell-known in the art.

All or the compositions given herein or claimed as part of a newinvention include the homologues of that composition. A homologue refersto a natural protein, encoded by a DNA molecule from the same or aprotein. Homologues, as used herein, typically share about 50% sequencesimilarity at the DNA level or about 18% sequence similarity in theamino acid sequence. Homologues are more aptly quantitated in thestatistical programs given below. An example a homologue of a nativestaphylococcal enterotoxin would be any structure including allsubstitution, deletion or addition mutants, derivatives, fusionproteins, chimeric proteins, fragments, conjugates, synthetic andnaturally occurring structures with a Z value >10 in the Lipman-PearsonFASTA/FASTP program.

The recognition that the biologically active regions of theenterotoxins, for example, are substantially structurally homologousenables predicting the sequence of synthetic peptides which exhibitsimilar biological effects in accordance with this invention (Johnson,L. P. et al., Mol. Gen. Genet. 203:354-356 (1886).

A common method for evaluating sequence homology, and more importantly,for identifying statistically significant similarities of the proteins,peptides and nucleic acids given herein is by Monte Carlo analysis usingan algorithm written by Lipman and Pearson to obtain a Z value (FASTA).According to this analysis, Z>6 indicates probable significance, andZ>10 is considered to be statistically significant (Pearson, W. R. et.al., Proc. Natl. Acad Sci. USA, 85:2444-2448 (1988); Lipman, D. J. etal, Science 227:1435-1441 (1985)). Synthetic peptides corresponding tothe compositions and enterotoxins and all other molecules describedherein are characterized in that they are substantially homologous inamino acid sequence to an enterotoxin or other native molecule to whichit is being compared with statistically significant (Z>6) sequencehomology and similarity to include alignment of cysteine residues andsimilar hydropathy profiles.

The Lipman-Pearson FASTA program may be used to determine homology of agiven protein using the BLOSUM 50 or PAM 250 scoring matrix, gappenalties of −12 and −2 and the PIR or SwissPROT database. The resultsare expressed as Z values or E ( ) values. For the present database(2001), the Z>13 indicates statistical significance.

Most deletions and insertions, and substitutions according to thepresent invention are those which do not produce radical changes in thecharacteristics of the protein or peptide molecule. However, when it isdifficult to predict the exact effect of the substitution, deletion, orinsertion in advance of doing so, one skilled in the art will appreciatethat the effect will be evaluated by routine screening assays, forexample direct or competitive immunoassay or biological assay asdescribed herein. Modifications of such proteins or peptide propertiesas redox or thermal stability, hydro-phobicity, susceptibility toproteolytic degradation or the tendency to aggregate with carriers orinto multimers are assayed by methods well known to the ordinarilyskilled artisan.

In the present invention, functional derivatives or homologues ofproteins, peptides, enterotoxins or other related toxins and nucleicacids including fusion proteins, mutants (deletion and addition types),variants, conjugates with other proteins including but not limited toantibodies, F(ab′)₂, Fv or Fd fragments, receptors or receptor ligands,synthetic polypeptides and nucleic acids characterized by substantialstructural homology to staphylococcal enterotoxin A, enterotoxin B,enterotoxin C, enterotoxin D, enterotoxin E, enterotoxin F (TSST-1) andStreptococcal pyrogenic exotoxins A-H as well as the newer enterotoxins(SEG, SEH, SEI, SEJ, SEK, SEL, SEM), SETs 1-5 and non-enterotoxinsuperantigens (e.g., Yersinia pseudotuberculosis superantigen,Mycoplasma arthitidis superantigen) with statistically significantsequence homology and similarity (e.g., Z>6 in the Lipman and Pearsonalgorithm in Monte Carlo analysis (FASTA program) or the preferredmethodology for determining sequence similarity and identity of proteinsand nucleic acid as given above in this section (e.g., ALIGN, NBLAST,XBLAST programs as described above) are included in the invention. Allof the superantigen conjugates to other polypeptides, peptides (e.g.,verotoxins, chemokine receptors, costimulants, invasins, viral antigens)given in this application are considered to be structural homologues areincluded in this invention as structural homologues if they show Zvalues >10 or additional statistical criteria for inclusion as given inthis section.

9. New Streptococcal Pyrogenic Exotoxins, Staphylococcal Enterotoxinsand SETs for Tumor Therapy

Streptococcal pyrogenic exotoxins SPEA, SPEB, SPEC, SPEC, SPEH, SPEHSME-Z, SME-Z₂ and SSA are superantigens induce tumoricidal effects.SPEG, SPEH, and SPEJ genes were identified from the Streptococcuspyogenes Ml genomic database at the University of Oklahoma. A fourthnovel gene (smez-2) was isolated from the S. pyogenes strain 2035, basedon sequence homology to the streptococcal mitogenic exotoxin z (smez)gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ andstreptococcal pyrogenic exotoxin (SPE)-C, whereas SPE-His most similarto the staphylococcal toxins than to any other streptococcal toxin.Recombinant (r)SMEZ, rSMEZ-2, rSPE-G, and rSPE-H were mitogenic forhuman peripheral blood lymphocytes. SMEZ-2 is the most potentsuperantigen (SAg) discovered thus far. All toxins, except rSPE-G, wereactive on murine T cells, but with reduced potency. Binding to a humanB-lymphoblastoid line was shown to be zinc dependent with high bindingaffinity of 15-65 nM. Evidence from modeled protein structures andcompetitive binding experiments suggest that high affinity binding ofeach toxin is to the major histocompatibility complex class II (3 chain.Competition for binding between toxins was varied and revealedoverlapping but discrete binding to subsets of class II molecules in thehierarchical order (SMEZ, SPE-C)>SMEZ-2>SPE-H>SPE-G. The most commontargets for these SAgs were human Vβ 2.1- and Vβ4-expressing T cells.

There are four naturally occurring SPEA alleles, and three of these,SPEA1, SPEA2, and SPEA3, encode toxins differing by a single amino acid.The toxin encoded by SPEA4 is 9% divergent from the other three, with 26amino acid changes. Twenty mutant forms of SPEA1 (SPEA encoded by allele1), and the mutant toxins were analyzed for mitogenic stimulation ofhuman peripheral blood mononuclear cells, affinity for class II majorhistocompatibility complex molecules (DQ), and disulfide bond formation.Residues necessary for each of these functions were Identified., Theproduct of allele 2, SPEA2, had slightly higher affinity for the classII MHC molecule compared with SPEA1 but not significantly greatermitogenic activity. SPEA3, however, was significantly increased inmitogenic activity and affinity for class II MHC compared with SPEA1.Thus, there is evidence that the toxin encoded by some of the highlyvirulent S. pyogenes STSS-associated isolates is a more active form ofSPEA.

A new ˜28-kDa superantigen protein designated streptococcal superantigen(SSA), was isolated from culture supernatants. SSA stimulatedproliferation of human T cells bearing Vβ1, Vβ3, Vβ5.2, and Vβ15 in anMHC class II-dependent manner. N-terminal sequencing found the first 24residues of SSA to be 62.5% identical to staphylococcal superantigensSEB, SEC1, and SEC3.

Newer Staphylococcal enterotoxins SEG, SEH, SEI, SEJ, SEK, SEL, SEM,ENT, ENT1, ENT2, also show superantigenic activity and are capable ofinducing tumoricidal effects. The homology of these toxins to othertoxins in the family ranges from 27-64%. Selective expansion of TCR Vβsubsets has been demonstrated for each. (Jarraud S et al., J. Immunol.166: 669-677 (2001); Jarraud S et al., J. Clin. Microbiol. 37: 2446-2449(1999); Munson, S H et al., Infect. Immun. 66:3337-3345 (1998 The abovestreptococcal and newer enterotoxins retain T cell activation and Vβusage. SPEA, SPEC, SPEG, SPEH, SME-Z, SME-Z₂, SEG, SEH, are known toutilize zinc as part of high affinity MHC class II receptor. Amino acidsubstitution(s) at the high affinity zinc dependent class II bindingsite is used to reduce their affinity for MHC class II receptor. Tumorlocalization is insured by the fusion with tumor specific antibodies,F(ab′)₂ or single chain Fv fragments. Additional or alternative tumorlocalizing motifs may be added to the toxin molecules which include butare not limited to an RGD motif, VEGF (localizing to KDR tyrosine kinasereceptors on vascular endothelium) and other tumor receptor ligands.

These proteins and their homologues are isolated and characterized as inExample 10 and. These proteins and their homologues are useful aspreventative or therapeutic antitumor vaccines according to Examples 5and 6 and in nucleic acid form as in Example 1.

10. Coaguligands: SEs Fused to Coagulation Factors

Superantigens may be conjugated to, or operatively associated with,polypeptides that are capable of directly or indirectly stimulatingcoagulation, thus forming a “coaguligand” (Barinaga M et al, Science275:482-4 (1997); Huang X et al, Science 275:547-50 (1997); Ran S et al,Cancer Res Oct. 15 1998; 58(20):4646-53; Gottstein C et al.,Biotechniques 30:190-4 (2001)).

In coaguligands, the antibody may be directly linked to a direct orindirect coagulation factor, or may be linked to a second binding regionthat binds and then releases a direct or indirect coagulation factor.The ‘second binding region’ approach generally uses a coagulant-bindingantibody as a second binding region, thus resulting in a bispecificantibody construct. The preparation and use of bispecific antibodies ingeneral is well known in the art, and is further disclosed herein.

Coaguligands are prepared by recombinant expression. The nucleic acidsequences encoding the SAg are linked, in-frame, to nucleic acidsequences encoding the chosen coagulant, to create an expression unit orvector. Recombinant expression results in translation of the new nucleicacid, to yield the desired protein product.

Where coagulation factors are used in connection with the presentinvention, any covalent linkage to the SAg should be made at a sitedistinct from the functional coagulating site. The compositions are thus“linked” in any operative manner that allows each region to perform itsintended function without significant impairment. Thus, the SAg binds toand stimulates T cells, and the coagulation factor promotes bloodclotting.

Preferred coagulation factors are Tissue Factor (“TF) compositions, suchas truncated TF (tTF), dimeric, multimeric and mutant TF molecules.“Truncated TF” (tTF) refers to TF constructs that are renderedmembrane-binding deficient by removal of sufficient amino acid sequencesto effect this change in property. A “sufficient amount” in this contextis an amount of transmembrane amino acid sequence originally sufficientto enter the TF molecule in the membrane, or otherwise mediatefunctional membrane binding of the TF protein. The removal of such a“sufficient amount of transmembrane spanning sequence” therefore createsa truncated TF protein or polypeptide deficient in phospholipid membranebinding capacity, such that the protein is substantially a solubleprotein that does not significantly bind to phospholipid membranes.Truncated TF thus substantially fails to convert Factor VII to FactorVila in a standard TF assay, and yet retains so-called catalyticactivity including activating Factor X in the presence of Factor Vila.

U.S. Pat. No. 5,504,067 is specifically incorporated herein by referencedescribes truncated TF proteins. Preferably, the TFs for use herein willgenerally lack the transmembrane and cytosolic regions (amino acids220-263) of the protein. However, there is no need for the truncated TFmolecules to be limited to molecules of the exact length of 219 aminoacids.

Any of the truncated, mutated or other TF constructs maybe prepared in adimeric form. TF dimers are prepared by employing the standardtechniques of molecular biology and recombinant expression, in which twocoding regions are prepared in-frame and expressed from an expressionvector. Equally, various chemical conjugation technologies may beemployed to prepare TF dimers. The individual TF monomers may bederivatized prior to conjugation.

The tTF constructs may be multimeric or polymeric. A “polymericconstruct” contains 3 or more Tissue Factor constructs. A “multimeric orpolymeric TF construct” is a construct that comprises a first TFmolecule or derivative operatively attached to at least a second and athird TF molecule or derivative. The multimers may comprise betweenabout 3 and about 20 such TF molecules. The constructs may be readilymade using either recombinant manipulation and expression or usingstandard synthetic chemistry.

TF mutants deficient in the ability to activate Factor VII are useful.Such “Factor VII activation mutants” are generally defined herein as TFmutants that bind functional Factor VH/VIIa, proteolytically activateFactor X, but are substantially free from the ability to proteolyticallyactivate Factor VII. Accordingly, such constructs are TF mutants thatlack Factor VII activation activity.

The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon their specificdelivery to the tumor vasculature, and the presence of Factor Vila atlow levels in plasma. Upon administration of such a Factor VIIactivation mutant conjugate, the mutant will be localized within thevasculature of a vascularized tumor. Prior to localization, the TFmutant would be generally unable to promote coagulation in any otherbody sites, on the basis of its inability to convert Factor VII toFactor VIIa. However, upon localization and accumulation within thetumor region, the mutant will then encounter sufficient Factor Vila fromthe plasma in order to initiate the extrinsic coagulation pathway,leading to tumor-specific thrombosis. Exogenous Factor VIIa could alsobe administered to the patient.

Any one or more of a variety of Factor VII activation mutants may beprepared and used in connection with the present invention. The FactorVII activation region generally lies between about amino acid 157 andabout amino acid 167 of the TF molecule. Residues outside this regionmay also prove to be relevant to the Factor VII activating activity.Mutations are inserted into any one or more of the residues generallylocated between about amino acid 106 and about amino acid 209 of the TFsequence (WO 94/07515; WO 94/28017; each incorporated herein byreference).

A variety of other coagulation factors may be used in connection withthe present invention, as exemplified by the agents set forth below.Thrombin, Factor V/Va and derivatives, Factor VHI/VIIIa and derivatives,Factor IX/IXa and derivatives, Factor X/Xa and derivatives, FactorXl/XIa and derivatives, Factor XH/XIIa and derivatives, FactorXlll/XIIIa and derivatives, Factor X activator and Factor V activatormay be used in the present invention.

11. Chemotherapeutic Agents

A variety of chemotherapeutic agents may be used in the combinedtreatment methods disclosed herein. Chemotherapeutic agents contemplatedas exemplary include, e.g., cisplatin (CDDP), adriamycin, actinomycin,mitomycin, caminomycin, daunomycin, doxorubicin, tamoxifen, taxol,taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16),5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa,methotrexate, campothecin, actinomycin-D, mitomycin C, aminopterin,combretastatin(s) and derivatives and prodrugs thereof. Anti-cancerchemotherapeutic drugs useful in this invention include but are notlimited to antimetabolites, anthracycline, vinca alkaloid, anti-tubulindrug, antibiotic, alkylating agent The chemotherapeutic agent(s)selected for use preferably shows the highest response rate againsttumor to be treated. For example, in non-small cell lung cancer, thecisplatin-based trials showed a benefit of chemotherapy with a hazardratio of 0.73 (p<0.0001), equivalent to an absolute improvement insurvival of 10% (5-15%) at 1 year, or an increase in median survival of1½ months (1-2½ months). Completed prospective randomized trialsincluding quality-of-life analyses show that cisplatin-based therapeuticregimens also improve quality of life in these patients. Other agents inphase III trials in patients with advanced NSCLC include the taxanes(paclitaxel and docetaxel), vinca alkaloid (vinorelbine), antimetabolite(gemcitabine), and campothecin (irinotecan). These agents have shownpromise in both phase I and II trials, both as single agents and incombination with a platinum agent.

EXAMPLES Example 1

Preparation of Plasmids for Making DNA Templates for any Gene ofInterest and the Process of Transfection

Mammalian oncogenes, and genes for oncogenic transcription factors,angiogenic factors, growth factor receptors and amplicons as well asbacterial and SAg plasmids and DNA are prepared as described in the textreferences. When necessary, they are modified to forms suitable fortransfection into mammalian tumor cells or accessory cells using methodswell described in the art. (Old R W et al., Principles of GeneManipulation, 5th Ed., Blackwell 1994).

As a representative SAg, enterotoxin B plasmid DNA is prepared by themethod of Jones C L et al., J. Bacteriology 166 29-33 (1986) and Ranelliet al., Proc. Natl. Acad. Sci. USA 82:5850-5854 (1985) using theCsCl-ethidium bromide density gradient centrifugation of cleared lysatesas described (Clewell, D B et al., Proc. Natl. Acad. Sci. USA62-1159-1166 (1969)). S. aureus chromosomal DNA was isolated asdescribed by Betley M et al., Proc. Natl. Acad. Sci. USA 81: 5179-5183(1984). E. coli HB101 was transformed with plasmid DNA by the CaCl₂procedure of Morrison D A et al., Meth. Enzymol. 68:326-331 (1979).Restriction digests were analyzed by 1% agarose and 5% acrylamide gelelectrophoresis using Tris/Borate/EDTA buffer as described in Greene P Jet al., Methods Mol. Biol. 7: 87-111 (1974). Additional methods forisolation and cloning of specific bacterial and mammalian plasmid DNAuseful in tumor or accessory cell transfection are cited in referencesgiven previously in the text or in Snyder L et al., Molecular Geneticsof Bacteria, ASM Press, Washington, D.C. (1997); Peters et al., supra;Franks et al., supra.

Suitable template DNA for production of mRNA encoding a desiredpolypeptide may be prepared using standard recombinant DNA methodologyas described in Ausubel F et al. Short Protocols in Molecular Biology3rd Ed. John Wiley, New York, N.Y. (1995). There are numerous availablecloning vectors and any cDNA containing an initiation codon can beintroduced into the selected plasmid and mRNA can be prepared from theresulting template DNA. The plasmid can be cut with an appropriaterestriction enzyme to insert any desired cDNA coding for a polypeptideof interest. For example the readily available cloning vector pSP64T canbe used after linearization and transcription with SP6 RNA polymerase.Smaller sequence may be inserted into the Hind III/EcoTI fragment withT4 ligase. Resulting plasmids are screened for orientation andtransformed into E. coli. These plasmids are adapted to receive any geneof interest at a unique BglII restriction site which is placed betweenthe two Xenopus β-globin sequences.

Subcloning of SEB into pHb-Apr-1-Neo Expression Vector:

The Staphylococcal enterotoxin B (SEB) gene has been subcloned intopHβ-Apr-1-neo expression vector. The final construct contained only thecoding sequence of SEB and conferred resistance to ampicillin and G-418.

Materials and Methods

PCR:

1. The following two primers are designed and made at Life Technologies,Inc.:

Primer SEB1: total 24 bp 5′ to 3′ GGC.GTC.GAC.ATG.TAT.AAG.AGA.TTASalI site:

Primer SEB2: total 24 bp 5′ to 3′ GCC.GGA.TCC.TCA.CTT.TTT.CTT.TGTBamHI site:Both primers were dissolved in filter-sterilized ddH₂O to a finalconcentration of 20 mM (stock solution).2. The volume (in ml) of reagents for each PCR reaction is listed below:

Exp. Exp. Reagent 1 2 Exp. 3 Exp. 4 Exp. 5 ddH₂O 76 72 67 49 59 10 X PCRbuffer 10 10 10 10 10 10 X dNTP (2 mM stock) 10 10 10 10 10 Primer SEB1(20 mM stock) 1 5 1 10 10 Primer SEB2 (20 mM stock) 1 1 1 10 10 SEBTemplate (50 mg stock) 1 1 10 10 0 PfuTurbo Enz 1 1 1 1 1 Final Volume100 100 100 100 1003. The following cycling parameters were applied:

95° C. 1 minute  1 cycle initial denature 95° C. 45 seconds denature 52°C. 1 minute 20 cycles anneal 72° C. 1 minute extension 72° C. 1 minute 1 cycle final extension  4° C. hold4. To verify that the PCR reactions yielded the correct size fragment,10 ml of the reaction mixture was electrophoresed on a 1% agarose gel in1×TAE buffer.Vector:1. The pHb-Apr-1-neo expression vector was spotted on a filter paper.2. To recover the DNA, the circle was cut out and added to 100 ml of H₂Oto allow rehydration for 5 minutes. After a brief centrifugation, thesupernatant was used to transform E. coli XL1Blue (Stratagene), andselected by ampicillin (final concentration 100 mg/ml).3. To verify that the vector is correct, 4 ampR clones were randomlyselected and the clones were cultured in LB amp media. DNA was isolatedand digested with SalI, BamHI (single digest) and EcoRI/HindIII (doubledigest). The digested products were electrophoresed on a 1% agarose gelin 1×TAE buffer. The profile of the restriction digest confirmed thatthe vector is correct.Cloning and Verification:1. The correct PCR fragments in experiments 2, 3, and 4 were pooled andgel-purified. A portion of the fragments was digested with restrictionenzymes SalI and BamHI, and was ligated into the digested pHb-Apr-1-neoexpression vector. The ligation products were transformed into E. coliXL1Blue (Stratagene). Insert containing clones were selected byampicillin.2. Ten ampicillin resistant clones were randomly selected, cultured in 5ml of LB amp media, and their plasmid DNA was isolated. Insertcontaining clones (SEB construct were verified by digesting the DNA withSalI and BamHI restriction endonucleases and electrophoresis at 0.8%agarose gel.3. One of the SEB constructs (clone #2) was verified by sequencing andaligned with the published SEB sequence. Purified DNA templates frombacteria and human cells are prepared for introduction of plasmid intohuman and bacterial cells by additional methods given in Ausubel F etal., supra. The plasmid DNA is grown up in E. coli in ampicillincontaining LB medium. The cells were then pelleted by spinning a 5000rpm for 10 min. at 5000 rpm., resuspended in cold TE pH 8.0, centrifugedagain for 10 minutes. at 5000 rpm., resuspended in a solution of 50 mMglucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA and 40 mg/ml lysozyme. Afterincubation for 5-10 min. with occasional inversion, 0.2 N NaOHcontaining 1% SDS was added, followed after 10 minutes at 0° C. with 3 Mpotassium acetate and 2 M acetic acid. After 10 more minutes, thematerial was again centrifuged a 6000 rpm, and the supernatant wasremoved with a pipet. The pellet was then mixed into 0.6 vol.isopropanol (−20° C.), mixed, and stored at −20° C. for 15 minutes. Thematerial was then centrifuged again at 10,000 rpm for 20 min., this timein an HB4 singing bucket rotor apparatus after which the supernatant wasremoved and the pellet was washed in 70% EtOH and dried at roomtemperature. Next, the pellet was resuspended in 3.5 ml TE, followed byaddition of 3.4 g CsCl and 3501 of 5 mg/ml EtBr. The resulting materialwas placed in a quick seal tube, filled to the top with mineral oil. Thetube was spun for 3.5 hours at 80,000 rpm in a VTi80 centrifuge. Theband was removed and the material was centrifuged again making up thevolume with 0.95 g CsCl/ml and 0.1 ml or 5 mg/ml EtBr/ml in TE. The EtBrwas then extracted with an equal volume of TE saturated N-Butanol afteradding 3 volumes of TE to the band. Next, 2.5 vol. EtOH was added, andthe material was precipitated at −20° C. for 2 hours. The resultant DNAprecipitate is used as a DNA template.Transfection of B16F10 Melanoma Cells:

G418 sensitivity: B16F10 melanoma cells (B16s) were first tested forsensitivity to G418 which will be used as the selectable marker. At 400ug/mL G418, B16s did not survive, while 200 and 300 ug/mL allowed somesurvival.

Transfection:

Lipofectamine was used to produce stably transfected B16s. Theconditions for transfection were those described protocol provided byLife Technologies. B16s were plated at 4×10⁵ cells/well in 6 wellplates, using Murine Complete Medium (MCM) described in Report 2. Cellswere cultured overnight. Optimal density is 50-80% confluent and isusually achieved by 18-24 after seeding at 1-3×105 cells/well. DNAsources consisted of SEB-G418 resistance containing vector, vector DNAwith G418 resistance gene only, and control DNA from PSK401 (no G418resistance marker). DNA concentrations were determined for the SEBcontaining and control vectors.

DNA source A260 DNA (ug/ml) SEB 0.09 0.45 Vector only 0.13 0.65 PSK 4010.15 0.75

Lipofectamine solutions and DNA solutions were prepared in 12×75 mmtubes, using OPTI-MEM (Life Technologies 31985). DNA solutions containedapproximately 2 ug in 100 uL OPTI-MEM; the LIPOFECTAMINE Reagent wasdiluted by adding 6 or 12 uL to OPTI-MEM at a final volume of 100 uL.The solutions were mixed and held at room temperature for 30 minutes.Specific DNA and Lipofectamine conditions were as follows:

Plated cells were rinsed once with 2 ml/well OPTI-MEM. To the abovetubes, 0.8 mL OPTI-MEM. This mixture was then overlayed onto the washedcell monolayers according to the above well designations. Cells wereincubated for 5 hours at 37° C. in 5% CO2. Murine Complete Medium with20% FBS but no antibiotics was then added at 1 ml/well. Cultures wererefed with standard MCM, at 3 mL/well, after 24 hours. Three days aftertransfection, cells from each transfection condition were subcultured bysplitting the total cell suspension 90:10 into 150 mm plates (one platereceived 90% of the cell suspension, the other received the remaining10%).

G418 Selection:

All plates were refed at 6 days after transfection with mediumcontaining 400 ug/mL G418. Plates were refed every 2 to 3 days with G418containing medium until day 17 after transfection. No growth wasobserved in wells 1-4 as expected. Plates initiated with 90% of the cellsuspension and showing growth were harvested, frozen, and stored at −80°C.

Primary Subcloning:

Ten colonies were selected from each well for wells 5, 7, 9, and 11.Subcloning was accomplished by the use of cloning cylinders as follows:After seating the cylinder, medium was aspirated and the isolated colonywas washed once with 100 uL of warmed trypsin-EDTA. This was aspiratedand replaced with fresh tyrpsin-EDTA. After incubation at 37° C. for 2minutes, the cells were recovered by centrifugation and transferred to atube containing 1 ml MCM, then replated by addition of 20 uL of cellsuspension to 15 mL MCM with G418 in 150 mm plates. The remaining cellsuspension was plated into 24 well plates, 4 wells/clone and all plateswere maintained at 37° C., 5% CO2. The 6 well plates were used to assessSEB expression on the cell surface as described under Detection ofpositive clones.

Secondary and Tertiary Subcloning and Preparation of Frozen Stocks:

These and all subsequent procedures were performed by me. Secondarysubcloning was performed as above at 7 days after initiation of primarysubclones. One colony/plate was selected for further subcloning (a totalof 40 colonies). The cell suspension was prepared in a total volume of 1mL; 100 uL was replated into 100 mm plates containing 10 mL MCM withG418. The remaining cell suspension was plated in 96 well plates at100/well, 2 replicates for assay. The 96 well plate was used fordetection of intracellular expression of SEB described under Detectionof positive clones.

Primary subcloning plates were cultured one additional day, thenharvested, frozen, and stored at −80° C. These frozen stocks aredesignated primary subclones. Secondary subclones were refed after 4days. Of 40 secondary clones, 36 regrew. Tertiary subcloning wasperformed after 8 days and frozen stocks of secondary clones wereprepared after 9 days. Tertiary clones were refed after 3 days inculture and subcultured after 7 days in culture. Plates were harvested,cells were resuspended in a total of 1 mL, and replated by addition of100 μL of the cell suspension to 100 mm plates with 15 mL MCM or 100μL/well in a 96 well plate. Frozen stocks of tertiary clones wereprepared.

Generation of Conditioned Medium for Assay of Supernatants:

After 7 days, 100 mm plates of tertiary clones were again replated. Thistime, cell counts were performed and 4.5×10⁵ cells were plated in 12well plates, one well/clone. The remaining cell suspension was frozenand stored at −80° C. After 4 days in culture, supernatants wereharvested, stored at 4° C., and the cells were replated into 100 mmplates. Supernatants were obtained from the 100 mm plates after 7 daysin culture. Frozen stocks were also generated from these plates.

Development of ELISA with HRP Rabbit Anti-SEB

Final ELISA conditions were as follows:

Assay Plate ProBind (Falcon #3915) Capture Rabbit anti-SEB (ToxinTechnologies # LBI202), Antibody 10 ug/mL in PBS, 50 uL/well, 1 hr, RTWash 3X with 0.1% casein, 0.1% Tween 20 in PBS Blocking 1% casein inPBS, 250 uL/well, overnight, 4° C. Antigen Supernatant used neat or SEBdiluted in PBS, 50 μL/well, 2 hr, RT Wash As above Primary Ab HRP Rabbitanti-SEB (Toxin Technologies # LBC202), 1/300 in block buffer, 50μL/well, 2 hr, RT Substrate OPD, 2.5 mg/mL in citrate buffer, pH 5.0,0.03% H₂0₂, 100 μl/well, 15 min, RT Stop 4 M H2SO4, 100 μL/well Read-outOD 490 nmResults: SEB produced a dose response curve (linear range 60 fg-60pg/mL) and the background was very low. Vector only clones produced onlybackground signals. One SEB transfected clone produced a strong signal,three produced moderate signals, and one other produced a weak butdefinite signal.

OD 490 nm SEB+ Vector only 1 2 mean 1 2 mean 9.1 0.097 0.112 0.104 0.0790.102 0.091 9.2 0.127 0.123 0.125 0.081 0.076 0.078 9.3 0.109 0.1040.106 0.087 0.070 0.079 9.4 0.444 0.393 0.418 0.077 0.077 0.077 9.50.163 0.087 0.125 0.075 0.074 0.074 9.6 0.516 0.522 0.519 0.066 0.0640.065 9.7 0.087 0.091 0.089 0.096 0.084 0.090 9.8 0.386 0.450 0.4180.080 0.071 0.075 9.9 0.137 0.122 0.130 0.071 0.070 0.071 11.1 0.0830.075 0.079 0.068 0.078 0.073 11.2 1.847 1.802 1.824 0.063 0.076 0.07011.3 0.071 0.077 0.074 0.076 0.074 0.075 11.4 0.087 0.084 0.086 0.0830.085 0.084 11.5 0.161 0.220 0.191 0.092 0.086 0.089 11.8 0.221 0.1000.160 0.080 0.081 0.080 11.9 0.080 0.091 0.085 0.077 0.072 0.074 11.100.290 0.254 0.272 0.081 0.112 0.097 11.10 0.268 0.263 0.265 0.093 0.1140.103Based on the SEB standard curve, the following concentrations werederived.

Clone number (pg/ml) SEB 11.2 4.146 9.6 0.152 9.4 0.118 9.8 0.118 11.100.081

Cells are transfected ex vivo or in vivo and implanted in acancer-bearing host. These transfected cells are also used to stimulatehost lymphocytes ex vivo. Once activated, the lymphocytes areadministered to the host. The ex vivo or in vitro introduction of DNAinto cells is accomplished by methods that (1) form DNA precipitateswhich are internalized by the target cell; (2) create DNA-containingcomplexes with charge characteristics that are compatible with DNAuptake by a target cell; or (3) result in the transient formation ofpores in the plasma membrane of a target cell exposed to an electricpulse (these pores are of sufficient size to allow DNA to enter thetarget cell).

Generally, two factors determine the method used: the duration ofexpression required (i.e., transient versus stable expression) and thetype of cell to be transfected. The specific details of exemplaryprocedures are described herein. Transfections are carried out by wellestablished methods including calcium phosphate precipitations, DEAEDextran transfection, and electroporation.

Calcium Phosphate Precipitation

A commonly used ex vivo and in vitro method to transfer DNA intorecipient cells involves the co-precipitation of the DNA of interestwith calcium phosphate. With this technique, DNA enters the cell insufficient quantities such that the treated cells are transformed withrelatively high frequency. Using a variety of cell types, transfectionefficiencies of up to 10-3 have been obtained. This is the method ofchoice for the generation of stable transfectants.

Variations of the basic technique have been developed. If thetransfection involves the transfer of plasmid DNA, then high molecularweight genomic DNA isolated from a defined cell or tissue source can beincluded. The addition of such DNA, called carrier DNA, often increasesthe efficiency of transfection by the plasmid DNA. Upon arrival of theplasmid DNA/carrier DNA/calcium phosphate co-precipitate to the nucleusof the treated cell, the plasmid DNA integrates into the carrier DNA,often in the tandem array, and this assembly of plasmid and carrier DNA,called a transgenome, subsequently integrates into the chromosome of thehost cell.

Another procedural option is the addition of a chemical shock step tothe transfection protocol. Either dimethylsulfoxide or glycerol areappropriate. The optimal concentrations and lengths of treatment varyaccording to cell type. The use of these agents dramatically affect cellviability and can be optimized as described elsewhere [Chen and Okayama,Mol. Cell. Biol. 7:2745 (1987)]. Specifically, incubation of cells withthe co-precipitate is optimal at 35° C. in 2-4% CO₂ for 15-24 hours. Inaddition, circular DNA is more active than linear DNA and a finerprecipitate is obtained when the DNA concentration is between 20-30mg/ml in the precipitation mix.

It is noted that incubator temperature, CO₂ concentration, and DNAconcentration can be varied to obtain the desired result. In addition,the temperature and CO₂ concentrations described below are not optimalfor cell growth and should be maintained only temporarily.

Method:

Day 1: 1.3×10⁶ cells are seeded per 100-mm dish. Cells are about 75%confluent when used to seed the dishes.

Day 2: A large calcium phosphate cocktail mixture to transfect manyplates simultaneously is prepared. This protocol is given for 1 ml (or1×100-mm dish equivalent) of solution. These amounts are scaled up asnecessary, allowing for an appropriate amount of sample-transfer errors.Adherence to sterile technique is critical. Sterile reagents, tips, andtubes are used.1. Add 1-20 g DNA (1 mg/ml in sterile TE, 10 mM Tris-HCl 1 mM EDTA pH7.05) to 0.45 ml sterile H₂O, Note: First “sterilize” DNA by ethanolprecipitation with NaCl (0.1M final aqueous concentration) and 2× volume200% ethanol.2. Add 0.5 ml 2×HEPES buffered saline. Mix well.3. Add 50 ml of 2.5 M CaCl₂, vortex immediately.4. Allow the DNA mixture to sit undisturbed for 15-30 minutes at roomtemperature.5. Add 1 ml of the DNA transfection cocktail directly to the medium inthe 100-mm dish (plated with cells on day 1).6. Incubate the dishes containing the DNA precipitate for 16 hours at37° C. Remove the media containing the precipitate and add freshcomplete growth media.7. Allow the cells to incubate for 24 hours. Post-incubation, thecultures can be split for subsequent selection. Split cultures 1:5;however, to isolate individual colonies for further analysis, splitcultures 1:10 and 1:100.DEAE Dextran Transfection

Typically, DEAE dextran transfection is used to transiently transfectcells in culture. This method is highly efficient and the DNA/DEAEdextran mixture used for transfection is relatively easy to prepare. Forexample, this method yields transfection efficiencies of as high as 80percent. DNA introduced into cells with this method, however, appears toundergo mutations at a higher rate than that observed with calciumphosphate-mediated transfection.

Method:

Briefly, a DEAE dextran mixture is prepared and the DNA sample ofinterest is added, mixed, and then transferred to the cells in culture.

Day 1: Cells are seeded at a concentration of 2×10⁴ cells/cm2 in a totalvolume of 2 ml/well (1.92×10⁵ cells/well of a six-well cluster dish).Cells should be about 75% confluent when used to seed the dishes.

Day 2: Resuspend 0.5 ml DEAE Dextran in Tris-buffered saline (TBS).Final DEAE Dextran concentration should be about 0.04%. Observe cellmonolayers microscopically. Cells should appear about 60-70% confluentand well distributed. Bring all reagents to room temperature. Aspirateoff growth media and wash monolayer once with 3 ml of phosphate bufferedsaline (PBS), followed by one wash with 3 ml of TBS. Aspirate off TBSsolution and add 100-125 ml of the appropriate DNA/DEAE-Dextran/TBSmixture to the wells. Incubate dishes at room temperature inside alaminar flow hood. Rock the dishes every 5 minutes for 1 hour, makingsure the DNA solution covers the cells. After the 1-hour incubationperiod, aspirate off the DNA solution and wash once with 3 ml of TBSfollowed by 3 ml of PBS. Remove the PBS solution by aspiration andreplace with 2 ml of complete growth media containing 100 M chloroquine.Incubate the dishes in an incubator set at 37° C. and 5% CO₂ for 4hours. Remove the media containing chloroquine and replace with 2-3 mlof complete growth media (no chloroquine). Incubate the transfectedcells for 1-3 days, after which the cells will be ready for analysis.The exact incubation period depends on the intent of the transfection.Optimal expression typically occurs at 3 days post-transfection.Electroporation

Electroporation is a process whereby cells in suspension are mixed withthe DNA to be transferred. This cell/DNA mixture is subsequently exposedto a high-voltage electric field. This creates pores in the membranes oftreated cells that are large enough to allow the passage ofmacromolecules such as DNA into the cells. Such DNA molecules areultimately transported to the nucleus and a subset of these moleculesare integrated into the host genome. The reclosing of the membrane poresis both time and temperature dependent and thus is delayed by incubationat 0° C., thereby increasing the probability that the molecule ofinterest will enter the cell.

Electroporation appears to work on virtually every cell type. With thistechnique, the efficiency of nucleic acid transfer is high for bothtransient transfection and stable transfection. One important technicaldifference between electroporation and other competing technologies isthat the number of input cells required for electroporation isconsiderably higher.

Method:

1. Harvest exponentially growing cells such as tumor cells or accessorycells by trypsinization, pellet, and wash twice with electroporationbuffer (Kriegler, M. Gene Transfer and Expression, W.H. Freeman and Co.,New York, N.Y. (1991)).

2. Resuspend cells in electroporation buffer at a concentration of2-20×10⁶ cells/ml in an electroporation cuvette.

3. Add 5-25 mg of DNA that has been linearized to the cell suspension

4. Insert or connect the electroporation electrode according to themanufacturer's instructions and subject cell/DNA mixture to an electricfield (pulse).

5. Return cell/DNA mixture to ice and incubate for 5 minutes.

6. Plate cells in non-selective medium. Biochemical selection may becarried out 24-48 hours later.

Lipofectamine

In vitro cell transfections can be done in 12-well plates, using 3.0 gplasmid DNA and Lipofectamine (GIBCO BRL), at 37° C. for 4 hours. Aftertransfection, the cells are cultured in 2.0 ml complete medium for 48hours and the cells are harvested. The cells are then washed in PBS.Stably transfected Chinese hamster ovary (CHO) and B16 lines areisolated by selection in 1.0 mg/ml G418 (GIBCO BRL). Cells are grown andpassaged in medium containing G418 for 3-4 weeks Mock transfected celllines (cells transfected with vector only) are used as controls.

Viral Vectors

Recombinant viral vectors containing the nucleic acid of interest canalso be used to introduce nucleic acid into a cell ex vivo or in vitro.It is noted that viral vectors are also used to transfect cells in vivo.These viral vectors can be DNA viruses such as herpesviruses,adenoviruses, and vaccinia viruses or RNA viruses such as retroviruses.The method and materials required to produce and use these viral vectorsex vivo, in vitro, and in vivo are commonly known in the art and areused in the invention described herein (Sambrook, J. et al., supra).

Selection:

Regardless of the method used to transfect a particular cell type,stably transfected cells are identified as follows. The DNA of interestcontains a selectable marker. Typically, a selectable marker encodes apolypeptide that confers drug resistance and the DNA containing thisresistance conferring nucleic acid is transfected into the recipientcell. Post transfection, the treated cells are allowed to grow for aperiod of time (24-48) hours to allow for efficient expression of theselectable marker. After an appropriate incubation time, transfectedcells are treated with media containing the concentration of drugappropriate for the selective survival and expansion of the transfectedand now drug resistant cells.

Many drug as well as non-drug selection methods are known in the art andcan be used in the invention described herein. For example, a detaileddescription of currently available drug selection strategies is providedin Kriegler M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman and Co. New York, N.Y. pp. 103-107 (1991).

General Method:

Sixteen hours after transfection, the transfected/infected cells are fedwith fresh, non-selective media. Twenty-four to forty-eight hours later,the cultures are split to a 1:5 or greater dilution and plated indrug-containing media. It is noted that cells are not placed indrug-containing media immediately after transfection in order to allow asufficient amount of time for the drug resistance nucleic acid to beexpressed and thus confer the drug resistant phenotype. Cell culturesare re-fed with drug-containing media every three days, at which timecultures are examined under a microscope to determine the efficiency ofdrug selection.

Site-Directed Mutagenesis by Polymerase Chain Reaction:

Introduction of Restriction Endonuclease Sites by PCR

PCR is the preferred method for introducing any desired sequence changeinto the DNA. The basic protocol is as follows:

Materials:

DNA sample to be mutagenized, pUC19 plasmid b vector or similarhigh-copy number plasmid having M13 flanking primer 500 ng/ml (100pM/μl) flanking sequence primers incorporating the restriction enzymesite

TE buffer

10× amplification buffer

2 mM 4dNTP mix

500 ng/ml (100 pM/ml) M13 flanking sequence primers: forward (NEB) andreverse (NEB)

5 U/ml Taq DNA polymerase

Mineral oil

Chloroform

Buffered phenol

100% ethanol

Appropriate restriction endonucleases

500 ml microcentrifuge tube

Automated thermal cycler

-   1. Subclone DNA to be mutagenized into high-copy number vector using    restriction sites flanking the area to be mutated.-   2. Prepare template DNA by plasmid miniprep. Resuspend 100 ng in TE    buffer to 1 ng/ml final.-   3. Synthesize oligonucleotide primers and purify by denaturing    polyacrylamide gel electrophoresis. Resuspend oligonucleotides in    500 l TE buffer. Determine absorbance at A260 and adjust to 500    ng/ml.-   4. Combine the following in each of two 500 l microcentrifuge tubes,    adding oligonucleotides 1 and 2 to separate tubes:    10 ml (10 ng) template DNA    10 ml 10× amplification buffer    10 ml 2 mM 4dNTP mix    1 ml (500 ng) oligonucleotide 1 or 2 (100 pM final)    1 ml (500 ng) appropriate M 13 flanking sequence primer, forward or    reverse (100 pM final).    H₂O to 99.5 μl    0.5 ml Taq DNA polymerase (5 U/ml)    Overlay reaction with 100 ml mineral oil.-   5. Carry out PCR in an automated thermal cycler for 20 to 25 cycles    under the following conditions:    45 sec 93° C.    2 min 50° C.    2 min 72° C.    After last cycle, extend for an additional 10 min at 72° C.-   6. Analyze 41 by nondenaturing agarose or occurrence gel    electrophoresis to verify that the amplification has yielded the    predicted product.-   7. Remove mineral oil and extract once with chloroform to remove    remaining oil. Extract with buffered phenol and concentrate by    precipitation with 100% ethanol.-   8. Digest half the amplified DNA with the restriction endonucleases    for the flanking and introduced sites. Purify digested fragments on    a low gelling/melting agarose gel.-   9. Ligate and subclone both fragments into an appropriately digested    vector to obtain a recombinant plasmid containing a single DNA    fragment incorporating the new restriction site.-   10. Transform plasmid into E. coli. Prepare DNA by plasmid miniprep.-   11. Analyze amplified fragment portion of plasmid by DNA sequencing    to confirm the addition of the mutation.    Introduction of Point Mutation by PCR    Materials:    DNA sample to be mutagenized    Oligonucleotide primers incorporating the point mutation    Klenow fragment of E. coli DNA polymerase I    Appropriate restriction endonuclease    Procedure:    1. Prepare template DNA (steps 1 and 2 of Basic Protocol).    2. Synthesize and purify oligonucleotide primers (3 and 4).    3. Amplify template DNA (steps 4 and 5 of Basic Protocol 1). After    final extension step, add 5 U Klenow fragment and incubate 15 min at    30° C.).    4. Analyze and process reaction (steps 6 and 7 of Basic Protocol).    5. Digest half the amplified fragments with the restriction    endonucleases for the flanking sequences. Purify digested fragments    on a low gelling/melting agarose gel.    6. Subclone the two amplified fragments into an appropriately    digested vector by blunt-end ligation.    7. Carry out steps 10 and 11 of Basic Protocol.    Introduction of a Point Mutation by Sequential PCR    Steps:    1. Prepare the template DNA (steps 1 and 2 of Basic Protocol 1).    2. Synthesize and purify the oligosaccharide primers (5 and 6).    3. Amplify the template and generate blunt-end fragments (step 3 of    Basic Protocol).    4. Purify fragments by nondenaturing agarose gel electrophoresis.    Resuspend in TE buffer at 1 ng/ml.    5. Combine the following in 500 ml microcentrifuge tube:    10 ml (10 ng) each amplified fragment    1 ml (500 ng) each flanking sequence primer (each 100 pM final)    10 ml 10× amplification buffer    10 ml 2 mM 4dNTP mix    0.5 ml Taq DNA polymerase (5 U/ml)    Overlay with 100 ml mineral oil.    6. Carry out PCR for 20 to 25 cycles (step 5 of Basic Protocol 1).    Analyze and process the reaction mix (steps 6 and 7 of Basic    Protocol 1).    7. Digest cDNA fragment with appropriate restriction endonuclease    for the flanking sites. Purify fragment on a low gelling/melting    agarose gel. Subclone into an appropriately digested vector.    8. Carry out steps 10 and 11, Basic Protocol 1.    Genomic Targeting and Genetic Conversion in Cancer Therapy

A number of cellular transformations are due, in large part, to a singlebase mutation that alters the function of the expressed protein.Alterations in the DNA sequence of a gene involved in cell proliferationcan have a significant effect on the viability of particular cells.Thus, the capacity to modulate the base sequence of such a gene would bea useful tool for cancer therapeutics. An experimental strategy thatcenters around site-specific DNA base mutation or correction using aunique chimeric oligonucleotide has been developed. This chimericmolecule has demonstrated higher recombinogenic activities thanidentical oligonucleotides containing only DNA residues, both in vitroand in vivo. The chimeric molecule is designed to hybridize to a targetsite within the genome and induce a single base mismatch at the residuetargeted for mutation. The DNA structure created at this site isrecognized by the host cell's repair system which mediates thecorrection reaction. For example, the bcr-abl fusion gene, the productof a translocation between human chromosomes 9 and 22, and the cause ofchronic myelogenous leukemia (CML) can be targeted for gene correction.Fusion genes or mutations which abound in cancer cells are excellenttargets for correction especially if (1) they are unique and arerecognized by the immune system as dominant or subdominant epitopes, (2)they are a single copy target; (3) the DNA sequence of the fusion geneor mutation is unique. The goal of such experiments is to knock-out thefusion gene by changing an amino acid codon into a stop codon through achimeric directed DNA repair system.

Targeted Gene Correction of Episomal DNA in Mammalian Cells Mediated bya Chimeric RNA/DNA Oligonucleotide

An experimental strategy to facilitate correction of single-basemutations of episomal targets in mammalian cells has been developed. Themethod utilizes a chimeric oligonucleotide composed of a contiguousstretch of RNA and DNA residues in a duplex conformation with doublehairpin caps on the ends. The RNA/DNA sequence is designed to align withthe sequence of the mutant locus and to contain the desired nucleotidechange. Activity of the chimeric molecule in targeted correction is usedin a with the aim of correcting a point mutation in the gene encodingthe human liver/bone/kidney alkaline phosphatase. When the chimericmolecule is introduced into cells containing the mutant gene on anextrachromosomal plasmid, correction of the point mutation isaccomplished with a frequency approaching 30%. These results extend theusefulness of the oligonucleotide-based gene targeting approaches byincreasing specific targeting frequency.

The site directed mutagenesis is used to carry out using the chimericDNA/RNA structure which enables the construct to target tumor cells invivo and in vitro. Such targeting structures include target seekingmoieties and can in principle be any structure that is able to bind to acell surface structure or that binds via biospecific affinity. Thetarget seeking moiety is primarily a disease specific structure selectedamong hormones, antibodies, growth factors. The biospecific affinitycounterpart may include interleukins (especially interleukin-2)antibodies (full length antibody, Fab, F(ab′2), Fv, single chainantibody and any other antigen binding antibody fragments (such as Fab)directed to a cells surface epitope or more preferably towards thebinding epitope for the a specific antibody. They may also includepolypeptides binding to the constant domains of immunoglobulins (e.g.,protein A and G and L), lectins, streptavidin, biotin etc. The termantibodies comprises monoclonal as well as polyclonal preparations. Thetargeting moiety may also be directed toward unique structures on moreor less healthy cells that regulate or control the development of adisease. or ligands for specific receptors on tumor cells). Thetargeting structure may be a nucleic acid, lipid or carbohydrate andvariations thereof which target receptors on the diseased cell. Thetargeting is not confined to diseased cells but may include additionalnormal cells as well.

Example 2

Cells Transfected with Nucleic Acids Encoding SAgs

Cultured VX-2 carcinoma cells were shown to retain their tumorigenicactivity after implantation into New Zealand white rabbits. Progressivetumor outgrowth was observed over a 3 week period. Nucleic acid encodingSEB isolated and characterized by Gaskill et al, J. Biol. Chem. 263:6276(1988) and Ranelli et al., Proc. Natl. Acad. Sci. USA 82:5850 (1985)were used to transfect tissue cultured VX-2 carcinoma cells usingtransfection methodology described in Example 1. Transfectants wereselected using G418 and the survival of SEB-transfected VX-2 carcinomacells was observed. In additional experiments, attempts were made totransfect murine 205 and 207 tumor cells with nucleic acid encoding SEB(the kind gift from Dr. Saleem Khan) and Streptococcal pyrogenicexotoxin A (the kind gift of Dr. Joseph Ferretti). Successfultransfection of murine MCA 205 and B16 cells by nucleic acids encodingSEA and SEC2 was achieved shortly thereafter by integrating the SAg DNAinto several retroviral vectors (MFG NEO) containing a growth hormoneleader sequence under the control of a chick B-actin promoter (Krause JC et al., J. Hematotherapy 6: 41-51 (1997)). In addition, murine tumorsMCA 205 fibrosarcoma cells and a spontaneous mammary carcinoma cellswere successfully transfected with nucleic acids encoding SEB (providedby Dr. Saleem Khan) using the β-actin promoter. Transfected mammarycarcinoma cells induced T cell proliferation in vitro. To demonstratethe anti-tumor capacity of tumor cells transfected with nucleic acidencoding a SAg, these transfectants were injected i.p. into syngeneichosts with established mammary carcinomas. These transfectantsdemonstrated a capacity to reduce micrometastases of wild type mammarytumor in vivo assessed in a clonogenic lung metastases assay. Theanti-tumor effect produced by the SEB transfectants was enhancedsignificantly by the co-administration of tumor cells transfected withnucleic acids encoding the costimulating molecule B7-1.

Example 3

Pharmaceutical Compositions and their Manufacture

A preferred delivery system is the sickled erythrocyte containing thenucleic acids of choice a given in Example 6. The sickled erythrocytesundergo ABO and RH phenotyping to select compatible cells for delivery.The cells are delivered intravenously or intrarterially in a bloodvessel perfusing a specific tumor site or organ e.g. carotid artery,portal vein, femoral artery etc. over the same amount of time requiredfor the infusion of a conventional blood transfusion. The quantity ofcells to be administered in any one treatment would range from one tenthto one half of a full unit of blood. The treatments are generally givenevery three days for a total of twelve treatments. However, thetreatment schedule is flexible and may be given for a longer of shorterduration depending upon the patients response.

Example 4

General Procedures for Administering Constructs in Human Tumor Modelsand Human Patients

The constructs described herein are tested for therapeutic efficacy inseveral well established rodent models which are considered to be highlyrepresentative as described in “Protocols for Screening Chemical Agentsand Natural Products Against Animal Tumors and Other Biological Systems(Third Edition)”, Cancer Chemother. Reports, Part 3, 3: 1-112, which ishereby incorporated by reference in its entirety. Additional tumormodels of carcinoma and sarcoma originating from primary sites andprepared as established tumors at primary and/or metastatic sites areutilized to test further the efficacy of the constructs.

Example 5

General Procedures for Administering Tumor Cells or Sickled ErythrocytesTransduced with SAgs and SAg-Activated T or NKT Cells in Human TumorModels and Human Patients

A. Tumor Cells Transduced with SAg Nucleic Acids Alone or Cotransfectedwith Oncogenes or Nucleic Acids Encoding Potent Immunogens and BacterialProducts

In a representative protocol, using the B16 melanoma or A20 lymphoma orother models given above, 10⁵-10⁷ transfected tumor cells are implantedsubcutaneously and 1-6 months later 10⁵-10⁷ untransfected tumor cells,are implanted. In the case of tumor cells cotransfected with severaltherapeutic nucleic acids, controls are established consisting of groupstransfected with only one of the nucleic acids. These singletransfectants are administered on the same schedule as thecotransfectants and assessed for capacity to prevent or reverse tumorgrowth compared to positive controls receiving tumor alone. The animalsreceiving the SAg transfected tumor cells show no evidence of growth ofthe wild type tumor and prolonged survival compared to the controls inwhich there is 100% appearance of the tumors. The differences arestatistically significant.

SAg transfected tumor cells are also used to treat established tumors asfollows. Transfected tumor cells, 10⁵-10⁷ are given 3-10 days after theappearance of established tumors. Results show statistically significantarrest of tumor growth, prolongation of survival in treated animalscompared to untreated controls.B. SAg-Activated Effector T or NKT CellsEffector T or NKT cells are generated as described elsewhere and areinfused intravenously in doses of 10⁶-10⁸ into syngeneic hosts that havepulmonary metastatic lesions established by injecting tumor cellsintravenously 3 to 12 days earlier. Twenty days later, the animals aresacrificed and pulmonary metastases measured in treated animals comparedto untreated controls. Results show statistically significant reductionin total number of pulmonary nodules and prolonged survival in thetreated group compared to untreated controls.

Example 6

General Test Evaluation Procedures for Constructs and SAg ActivatedEffector T or NKT Cells

I. General Test Evaluation Procedures

A. Calculation of Mean Survival Time

Mean survival time is calculated according to the following formula:

${{Mean}\mspace{14mu}{survival}\mspace{14mu}{time}\mspace{14mu}({days})} = \frac{S + {{AS}\left( {}_{A - 1} \right)} - {\left( {B + 1} \right){NT}}}{{S\left( {}_{A - 1} \right)} - {NT}}$

DEFINITIONS

Day: Day on which deaths are no longer considered due to drug toxicity.Example: with treatment starting on Day 1 for survival systems (such asL1210, P388, B16, 3LL, and W256):

Day A: Day 6.

Day B: Day beyond which control group survivors are considered“no-takes.”

Example: with treatment starting on Day 1 for survival systems (such asL1210, P388, and W256), Day B-Day 18. For B16, transplanted AKR, and 3LLsurvival systems, Day B is to be established.

S: If there are “no-takes” in the treated group, S is the sum from Day Athrough Day B. If there are no “no-takes” in the treated group, S is thesum of daily survivors from Day A onward.

S_((A-1)): Number of survivors at the end of Day (A-1).

Example: for 3LE21, S_((A-1))=number of survivors on Day 5.

NT: Number of “no-takes” according to the criteria given in Protocols7.300 and 11.103.

B. T/C Computed for all Treated Groups

T/C is the ratio (expressed as a percent) of the mean survival time ofthe treated group divided by the mean survival time of the controlgroup. Treated group animals surviving beyond Day B, according to thechart below, are eliminated from calculations:

No. of survivors in Percent of “no-takes” treated group beyond Day B incontrol group Conclusion 1 Any percent “no-take” 2 <10 drug inhibition≧10 “no-takes” ≧3 <15 drug inhibitions ≧15 “no-takes”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of T/C for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as “cures” or “no-takes.”

Calculation of Median Survival Time

Median Survival Time is defined as the median day of death for a test orcontrol group. If deaths are arranged in chronological order ofoccurrence (assigning to survivors, on the final day of observation, a“day of death” equal to that day), the median day of death is a dayselected so that one half of the animals died earlier and the other halfdied later or survived. If the total number of animals is odd, themedian day of death is the day that the middle animal in thechronological arrangement died. If the total number of animals is even,the median is the arithmetical mean of the two middle values. Mediansurvival time is computed on the basis of the entire population andthere are no deletion of early deaths or survivors, with the followingexception:

C. Computation of Median Survival Time from Survivors

If the total number of animals including survivors (N) is even, themedian survival time (days) (X+Y)/2, where X is the earlier day when thenumber of survivors is N/2, and Y is the earliest day when the number ofsurvivors (N/2)-1. If N is odd, the median survival time (days) is X.

D. Computation of Median Survival Time from Mortality Distribution

If the total number of animals including survivors (N) is even, themedian survival time (days) (X+Y)/2, where X is the earliest day whenthe cumulative number of deaths is N/2, and Y is the earliest day whenthe cumulative number of deaths is (N/2)+1. If N is odd, the mediansurvival time (days) is X.

Cures and “No-Takes”: “Cures” and “no-takes” in systems evaluated bymedian survival time are based upon the day of evaluation. On the day ofevaluation any survivor not considered a “no-take” is recorded as a“cure.” Survivors on day of evaluation are recorded as “cures” or“no-takes,” but not eliminated from the calculation of the mediansurvival time.

E. Calculation of Approximate Tumor Weight from Measurement of TumorDiameters with Vernier Calipers

The use of diameter measurements (with Vernier calipers) for estimatingtreatment effectiveness on local tumor size permits retention of theanimals for lifespan observations. When the tumor is implanted sc, tumorweight is estimated from tumor diameter measurements as follows. Theresultant local tumor is considered a prolate ellipsoid with one longaxis and two short axes. The two short axes are assumed to be equal. Thelongest diameter (length) and the shortest diameter (width) are measuredwith Vernier calipers. Assuming specific gravity is approximately 1.0,and Pi is about 3, the mass (in mg) is calculated by multiplying thelength of the tumor by the width squared and dividing the product bytwo. Thus,

${{Tumor}\mspace{14mu}{weight}\mspace{14mu}({mg})} = {\frac{{length}\mspace{14mu}({mm}) \times \left( {{width}{\;\mspace{11mu}}\lbrack{mm}\rbrack} \right)^{2}}{2}\mspace{14mu}{Or}\mspace{14mu}\frac{L\; \times (W)^{2}}{2}}$

The reporting of tumor weights calculated in this way is acceptableinasmuch as the assumptions result in as much accuracy as theexperimental method warrants.

F. Calculation of Tumor Diameters

The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a T/C of 75% isapproximately equivalent to a tumor weight T/C of 42%.

G. Calculation of Mean Tumor Weight from Individual Excised Tumors

The mean tumor weight is defined as the sum of the weights of individualexcised tumors divided by the number of tumors. This calculation ismodified according to the rules listed below regarding “no-takes.” Smalltumors weighing 39 mg or less in control mice or 99 mg or less incontrol rats, are regarded as “no-takes” and eliminated from thecomputations. In treated groups, such tumors are defined as “no-takes”or as true drug inhibitions according to the following rules:

Percent of small tumors Percent of “no-takes” in treated group incontrol group Action ≦17 Any percent no-take; not used in calculations18-39 <10 drug inhibition; use in calculations ≧10 no-takes; not used incalculations ≧40 <15 drug inhibition; use in calculations ≧15 Code allnontoxic tests “33”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control. T/C are computed for all treated groupshaving more than 65% survivors. The T/C is the ratio (expressed as apercent) of the mean tumor weight for treated animals divided by themean tumor weight for control animals. SDs of the mean control tumorweight are computed the factors in a table designed to estimate SD usingthe estimating factor for SD given the range (difference between highestand lowest observation). Biometrik Tables for Statisticians (Pearson ES, and Hartley H G, eds.) Cambridge Press, vol. 1, table 22, p. 165.

II. Specific Tumor Models

A. Lymphoid Leukemia L1210

Summary: Ascitic fluid from donor mouse is transferred into recipientBDF₁ or CDF₁ mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is i.p., thecomposition being tested is administered i.p., and the parameter is meansurvival time. Origin of tumor line: induced in 1948 in spleen and lymphnodes of mice by painting skin with MCA. J Natl Cancer Inst. 13:1328(1953).Animals:Propagation: DBA/2 mice (or BDF₁ or CDF₁ for one generation).Testing: BDF₁ (C57BL/6×DBA/2) or CDF₁ (BALB/c×DBA/2) mice.Weight: Within a 3-g weight range, with a minimum weight of 18 g formales and 17 g for females.Sex: One sex used for all test and control animals in one experiment.Experiment Size Six animals per test group.Control Groups Number of animals varies according to number of testgroups.Tumor Transfer:Inject i.p., 0.1 ml of diluted ascitic fluid containing 10⁵ cells.Time of Transfer for Propagation: Day 6 or 7.Time of Transfer for Testing: Day 6 or 7.Testing ScheduleDay 0: Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily.Day 1: Weigh and randomize animals. Begin treatment with therapeuticcomposition. Typically, mice receive 1 ug of the test composition in 0.5ml saline. Controls receive saline alone. The treatment is given as onedose per week. Any surviving mice are sacrificed after 4 weeks oftherapy.Day 5: Weigh animals and record.Day 20: If there are no survivors except those treated with positivecontrol compound, evaluate study.Day 30: Kill all survivors and evaluate experiment.Quality Control

Acceptable control survival time is 8-10 days. Positive control compoundis 5-fluorouracil; single dose is 200 mg/kg/injection, intermittent doseis 60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. Ratio oftumor to control (T/C) lower limit for positive control compound is 135%

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a composition shouldhave two multi-dose assays that produce a T/C 25%.

B. Lymphocytic Leukemia P388

Summary: Ascitic fluid from donor mouse is implanted in recipient BDF₁or CDF₁ mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is ip, thecomposition being tested is administered ip daily for 9 days, and theparameter is median survival time. Origin of tumor line: induced in 1955in a DBA/2 mouse by painting with MCA. Scientific Proceedings,Pathologists and Bacteriologists 33:603, 1957.Animals:Propagation: DBA/2 mice (or BDF₁ or CDF₁ for one generation)Testing: BDF₁ (C57BL/6×DBA/2) or CDF₁ (BALB/c×DBA/2) mice.Weight: Within a 3-g weight range, with a minimum weight of 18 g formales and 17 g for females.Sex: One sex used for all test and control animals in one experiment.Experiment Size: Six animals per test group.Control Groups Number of animals varies according to number of testgroups.Tumor TransferImplant: Inject ipSize of Implant: 0.1 ml diluted ascitic fluid containing 10⁶ cells.Time of Transfer for Propagation: Day 7.Time of Transfer for Testing: Day 6 or 7.Testing ScheduleDay 0: Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily.Day 1: Weigh and randomize animals. Begin treatment with therapeuticcomposition. Typically, mice receive 1 ug of the composition beingtested in 0.5 ml saline. Controls receive saline alone. The treatment isgiven as one dose per week. Any surviving mice are sacrificed after 4weeks of therapy.Day 5: Weigh animals and record.Day 20: If there are no survivors except those treated with positivecontrol compound, evaluate experiment.Day 30: Kill all survivors and evaluate experiment.Quality Control

Acceptable median survival time is 9-14 days. Positive control compoundis 5-fluorouracil: single dose is 200 mg/kg/injection, intermittent doseis 60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. T/C lowerlimit for positive control compound is 135% Check control deaths, notakes, etc.

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a synthetic must havetwo multi-dose assays (each performed at a different laboratory) thatproduce a T/C 125%; a natural product must have two different samplesthat produce a T/C 125% in multi-dose assays.

C. Melanotic Melanoma B 16

Summary: Tumor homogenate is implanted ip or sc in BDF₁ mice. Treatmentbegins 24 hours after either ip or sc implant or is delayed until an sctumor of specified size (usually approximately 400 mg) can be palpated.Results expressed as a percentage of control survival time. Thecomposition being tested is administered ip, and the parameter is meansurvival time. Origin of tumor line: arose spontaneously in 1954 on theskin at the base of the ear in a C57BL/6 mouse. Handbook on GeneticallyStandardized Jax Mice. Roscoe B. Jackson Memorial Laboratory, BarHarbor, Me., 1962. See also Ann NY Acad Sci 100, Parts 1 and 2, 1963.Animals:Propagation: C57BL/6 mice.Testing: BDF₁ (C57BL/6×DBA/2) mice.Weight: Within a 3-g weight range, with a minimum weight of 18 g formales and 17 g for females.Sex: One sex used for all test and control animals in one experiment.Experiment Size Ten animals per test group. For control groups, thenumber of animals varies according to number of test groups.Tumor TransferPropagation: Implant fragment sc by trochar or 12-gauge needle or tumorhomogenate (see below) every 10-14 days into axillary region withpuncture in inguinal region.Testing: Excise sc tumor on Day 10-14.Homogenate: Mix 1 g or tumor with 10 ml of cold balanced salt solutionand homogenize, and implant 0.5 ml of this tumor homogenate ip or sc.Fragment: A 25-mg fragment may be implanted sc.Testing ScheduleDay 0: Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily.Day 1: Weigh and randomize animals. Begin treatment with therapeuticcomposition. Typically, mice receive 1 μg of the composition beingtested in 0.5 ml saline. Controls receive saline alone. The treatment isgiven as one dose per week. Any surviving mice are sacrificed 8 weeks oftherapy.Day 5: Weigh animals and record.Day 60: Kill all survivors and evaluate experiment.Quality Control

Acceptable control survival time is 14-22 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. T/C lower limit for positive control compound is 135%Check control deaths, no takes, etc.

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a therapeuticcomposition should have two multi-dose assays that produce a T/C 125%.

Metastasis after IV Injection of Tumor Cells

10⁵ B16 melanoma cells in 0.3 ml saline are injected intravenously inC57BL/6 mice. The mice are treated intravenously with 1 g of thecomposition being tested in 0.5 ml saline. Controls receive salinealone. The treatment is given as one dose per week. Mice sacrificedafter 4 weeks of therapy, the lungs are removed and metastases areenumerated.

C. 3LL Lewis Lung Carcinoma

Summary: Tumor may be implanted sc as a 2-4 mm fragment, or im as a2×10⁶-cell inoculum. Treatment begins 24 hours after implant or isdelayed until a tumor of specified size (usually approximately 400 mg)can be palpated. The composition being tested is administered ip dailyfor 11 days and the results are expressed as a percentage of thecontrol.Origin of tumor line: arose spontaneously in 1951 as carcinoma of thelung in a C57BL/6 mouse. Cancer Res 15:39, 1955. See, also Malave, I. etal., J. Nat'l. Canc. Inst. 62:83-88 (1979).Animals:Propagation: C57BL/6 mice.Testing: BDF₁ mice or C3H.Weight: Within a 3-g weight range, with a minimum weight of 18 g formales and 17 g for females.Sex: One sex used for all test and control animals in one experiment.Experiment Size Six animals per test group for sc implant, or ten for imimplant. For control groups, the number of animals varies according tonumber of test groups.Tumor TransferImplant: Inject cells im in hind leg or implant fragment sc in axillaryregion with puncture in inguinal region.Time of Transfer for Propagation: Days 12-14.Time of Transfer for Testing: Days 12-14.Testing ScheduleDay 0: Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily.Day 1: Weigh and randomize animals. Begin treatment with therapeuticcomposition. Typically, mice receive 1 ug of the composition beingtested in 0.5 ml saline. Controls receive saline alone. The treatment isgiven as one dose per week. Any surviving mice are sacrificed after 4weeks of therapy.Day 5: Weigh animals and record.Final Day: Kill all survivors and evaluate experiment.Quality Control

Acceptable im tumor weight on Day 12 is 500-2500 mg. Acceptable im tumormedian survival time is 18-28 days. Positive control compound iscyclophosphamide: 20 mg/kg/injection, qd, Days 1-11. Check controldeaths, no takes, etc.

Evaluation

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C 125%is considered necessary to demonstrate activity. For confirmed activitya synthetic must have two multi-dose assays (each performed at adifferent laboratory); a natural product must have two differentsamples.

D. 3LL Lewis Lung Carcinoma Metastasis Model

This model has been utilized by a number of investigators. See, forexample, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264 (1980);Gorelik, E. et al., Rec. Results Canc. Res. 75:20-28 (1980); Isakov, N.et al., Invasion Metas. 2:12-32 (1982) Talmadge J. E. et al., J. Nat'l.Canc. Inst. 69:975-980 (1982); Hilgard, P. et al., Br. J. Cancer35:78-86 (1977)).

Mice: male C57BL/6 mice, 2-3 months old.

Tumor: The 3LL Lewis Lung Carcinoma was maintained by sc transfers inC57BL/6 mice. Following sc, im or intra-footpad transplantation, thistumor produces metastases, preferentially in the lungs. Single-cellsuspensions are prepared from solid tumors by treating minced tumortissue with a solution of 0.3% trypsin. Cells are washed 3 times withPBS (pH 7.4) and suspended in PBS. Viability of the 3LL cells preparedin this way is generally about 95-99% (by trypan blue dye exclusion).Viable tumor cells (3×10⁴-5×10⁶) suspended in 0.05 ml PBS are injectedinto the right hind foot pads of C57BL/6 mice. The day of tumorappearance and the diameters of established tumors are measured bycaliper every two days.

Typically, mice receive 1 ug of the composition being tested in 0.5 mlsaline. Controls receive saline alone. The treatment is given as one ortwo doses per week. In experiments involving tumor excision, mice withtumors 8-10 mm in diameter are divided into two groups. In one group,legs with tumors are amputated after ligation above the knee joints.Mice in the second group are left intact as nonamputated tumor-bearingcontrols. Amputation of a tumor-free leg in a tumor-bearing mouse has noknown effect on subsequent metastasis, ruling out possible effects ofanesthesia, stress or surgery. Surgery is performed under Nembutalanesthesia (60 mg veterinary Nembutal per kg body weight).

Determination of Metastasis Spread and Growth

Mice are killed 10-14 days after amputation. Lungs are removed andweighed. Lungs are fixed in Bouin's solution and the number of visiblemetastases is recorded. The diameters of the metastases are alsomeasured using a binocular stereoscope equipped with amicrometer-containing ocular under 8× magnification. On the basis of therecorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur, M. L. et al.,J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 μg of FdUrd is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice. After 30 min, mice aregiven 1 μCi of ¹²⁵IdUrd. One day later, lungs and spleens are removedand weighed, and a degree of ¹²⁵IdUrd incorporation is measured using agamma counter.

Statistics: Values representing the incidence of metastases and theirgrowth in the lungs of tumor-bearing mice are not normally distributed.Therefore, non-parametric statistics such as the Mann-Whitney U-Test maybe used for analysis.

Study of this model by Gorelik et al. (1980, supra) showed that the sizeof the tumor cell inoculum determined the extent of metastatic growth.The rate of metastasis in the lungs of operated mice was different fromprimary tumor-bearing mice. Thus in the lungs of mice in which theprimary tumor had been induced by inoculation of large doses of 3LLcells (1-5×10⁶) followed by surgical removal, the number of metastaseswas lower than that in nonoperated tumor-bearing mice, though the volumeof metastases was higher than in the nonoperated controls. Using¹²⁵IdUrd incorporation as a measure of lung metastasis, no significantdifferences were found between the lungs of tumor-excised mice andtumor-bearing mice originally inoculated with 1×10⁶ 3LL cells.Amputation of tumors produced following inoculation of 1×10⁵ tumor cellsdramatically accelerated metastatic growth. These results were in accordwith the survival of mice after excision of local tumors. The phenomenonof acceleration of metastatic growth following excision of local tumorshad been observed by other investigators. The growth rate and incidenceof pulmonary metastasis were highest in mice inoculated with the lowestdoses (3×10⁴-1×10⁵ of tumor cells) and characterized also by the longestlatency periods before local tumor appearance. Immunosuppressionaccelerated metastatic growth, though nonimmunologic mechanismsparticipate in the control exerted by the local tumor on lung metastasisdevelopment. These observations have implications for the prognosis ofpatients who undergo cancer surgery.

E. Walker Carcinosarcoma 256

Summary: Tumor may be implanted sc in the axillary region as a 2-6 mmfragment, im in the thigh as a 0.2-ml inoculum of tumor homogenatecontaining 10⁶ viable cells, or ip as a 0.1-ml suspension containing 10⁶viable cells. Treatment of the composition being tested is usually ip.Origin of tumor line: arose spontaneously in 1928 in the region of themammary gland of a pregnant albino rat. J Natl Cancer Inst 13:1356,1953.Animals:Propagation: Random-bred albino Sprague-Dawley rats.Testing: Fischer 344 rats or random-bred albino rats.Weight Range: 50-70 g (maximum of 10-g weight range within eachexperiment).Sex: One sex used for all test and control animals in one experiment.Experiment Size Six animals per test group. For control groups, thenumber of animals varies according to number of test groups.Time of Tumor TransferTime of Transfer for Propagation: Day 7 for im or ip implant; Days 11-13for sc implant.Time of Transfer for Testing: Day 7 for im or ip implant; Days 11-13 forsc implant.Tumor TransferSc fragment implant is by trochar or 12-gauge needle into axillaryregion with puncture in inguinal area. Im implant is with 0.2 ml oftumor homogenate (containing 10⁶ viable cells) into the thigh. Ipimplant is with 0.1 ml of suspension (containing 10⁶ viable cells) intothe ip cavity.Testing SchedulePrepare and administer compositions under test on days, weigh animals,and evaluate test on the days listed in the following tables.

Test system Prepare drug Administer drug Weight animals Evaluate 5WA16 23-6 3 and 7  7 5WA12 0 1-5 1 and 5 10-14 5WA31 0 1-9 1 and 5 30Day 0: Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily.Day 1: Weigh and randomize animals.Final Day: Kill all survivors and evaluate experiment.Quality ControlAcceptable im tumor weight or survival time for the above three testsystems: 5WA16: 3-12 g. 5WA12: 3-12 g. 5WA31 or 5WA21: 5-9 days.Evaluation

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C 125%is considered necessary to demonstrate activity. For confirmed activitya therapeutic agent must have activity in two multi-dose assays.

F. A20 Lymphoma

10⁶ murine A20 lymphoma cells in 0.3 ml saline are injectedsubcutaneously in Balb/c mice. The mice are treated intravenously with 1g of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Tumor growthis monitored daily by physical measurement of tumor size and calculationof total tumor volume. After 4 weeks of therapy the mice are sacrificed.

Use in Established Tumors

For proteins or nucleic acid constructs, treatment consists of injectinganimals iv or ip with 50, 500 1000 or 5,000 ng of in 0.1-0.5 ml ofnormal saline. Unless indicated otherwise above, treatments are givenone to three times per week for two to five weeks. Phage displays areadministered as 10⁹ transducing units (TU) and irradiated bacterialcells as 10⁵ cells iv into the tail vein one to three times per week fortwo to five weeks. Exosomes or vesicles, harvested from transfected,transformed or fusion tumor cells or sickled cells are given i.v. intothe tail vein in a dose of 0.25-1 g per animal one to three times perweek for two to five weeks. The results shown in Table VI are for eachcomposition and dose tested. The results are statistically significantby the Wilcoxon rank sum test.

TABLE VI Tumor Model Parameter % of Control Response L1210 Mean survivaltime >130%  P388 Mean survival time >130%  B16 Mean survival time >130% B16 metastasis Median number of metastases <70% 3LL Mean survivaltime >130%  Mean tumor weight <40% 3LL metastasis Median survivaltime >130%  Mean lung weight <60 Median number of metastases <60% Medianvolume of metastases <60% Medial volume of metastases <60% Median uptakeof IdUrd <60% Walker carcinoma Median survival time >130%  Mean tumorweight <40% A20 Mean survival time >130%  Mean tumor volume <40%Antitumor Effects of Therapeutic Constructs and Effector T, NKT Cells orSickled Erythrocytes in Human Patients

All patients treated have histologically confirmed malignant diseaseincluding carcinomas, sarcomas, melanomas, lymphomas and leukemia andhave failed conventional therapy. Patients may be diagnosed as havingany stage of metastatic disease involving any organ system. Stagingdescribes both tumor and host, including organ of origin of the tumor,histologic type and histologic grade, extent of tumor size, site ofmetastases and functional status of the patient. A generalclassification includes the known ranges of Stage I (localized disease)to Stage 4 (widespread metastases). Patient history is obtained andphysical examination performed along with conventional tests ofcardiovascular and pulmonary function and appropriate radiologicprocedures. Histopathology is obtained to verify malignant disease.

Example 7

Treatment Procedures

Constructs (or Preparations)

Doses of the constructs are determined as described above using, interalia, appropriate animal models of tumors. Treatments are given 3×/weekfor a total of 12 treatments. Patients with stable or regressing diseaseare treated beyond the 12^(th) treatment. Treatment is given on eitheran outpatient or inpatient basis as needed.

Patient Evaluation

Assessment of response of the tumor to the therapy is made once per weekduring therapy and 30 days thereafter. Depending on the response totreatment, side effects, and the health status of the patient, treatmentis terminated or prolonged from the standard protocol given above. Tumorresponse criteria are those established by the International UnionAgainst Cancer and are listed in Table VII.

TABLE VII RESPONSE DEFINITION Complete remission (CR) Disappearance ofall evidence of disease Partial remission (PR) >50% decrease in theproduct of the two greatest perpendicular tumor diameters; no newlesions Less than partial remission 25-50% decrease in tumor size,stable for (<PR) at least 1 month Stable disease <25% reduction in tumorsize; no progression or new lesions Progression >25% increase in size ofany one measured lesion or appearance of new lesions despitestabilization or remission of disease in other measured sites

The efficacy of the therapy in a population is evaluated usingconventional statistical methods including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements can be evaluated separately.

Results

One hundred and fifty patients are treated. The results are summarizedin Table VIII. Positive tumor responses are observed in 80% of thepatients as follows:

TABLE VIII All Patients Response No. % PR 20 66% <PR 10 33% Tumor TypesResponse % of Patients Breast Adenocarcinoma PR + <PR 80%Gastrointestinal Carcinoma PR + <PR 75% Lung Carcinoma PR + <PR 75%Prostate Carcinoma PR + <PR 75% Lymphoma/Leukemia PR + <PR 75% Head andNeck Cancer PR + <PR 75% Renal and Bladder Cancer PR + <PR 75% MelanomaPR + <PR 75%

Example 8

Methods for Preparing Sickled Erythrocytes for Use as CarriersTumoricidal Agents

The sickled cells are obtained from patients with sickle cell anemia orsickle cell trait. The type of sickle cell disease may be hemoglobin SS,hemoglobin SC, or the combination of hemoglobin SS and β-thalassemia. Todetermine compatibility of donor sickled erythrocytes with recipienterythrocytes, the donor cells are ABO typed and matched. The tendency ofthese red cells to adhere to cultured endothelial cells is assayed invitro by the method of Hebbel R P et al., New Eng. J. Med. 302: 992-995(1980). The sickled cells are harvested, transfected with appropriateoncolytic or tumor specific viruses, toxins or anaerobic bacteria invitro by methods given in Example 1. Fifty to 250 cc of transfectedsickled erythrocytes is infused intravenously over 1-2 hours. Theprocedure is repeated two to three times weekly for two to four weeks.Responsive patients are retreated on a similar schedule if tumorreappears. The patient's vital signs are monitored every 10 minutesduring the infusion, then every hour for the next 4 hours and Q4-6 hoursthereafter.

Infection of nucleated erythrocytes by oncolytic or tumor specificviruses: This is carried out by the method of Muhlemann, O., Akusjarvi,G., in Adenovirus Methods and Protocols WSM Wold, editor, Humana Press,Totowa, N.J. (1999). Essential steps are given below. Transfection ofnucleated sickled cells with various plasmid DNAs described in section 6is carried out as in Example 1.

Infection of Sickled Cells with Adenovirus:

Sickled cells are grown in round cell-culture bottles on a magneticstirrer at 37° C. in MEM spinner cell medium, 5% newborn calf serum,optionally containing 1% penicillin/streptomycin. The cells must be keptin log phase (titer 2-6×10⁵ cells/mL), doubling time approx 24 h.

1. Start with 2-3×10⁹ sickled spinner cells, collect them bycentrifugation in sterile 1-L plastic bottles by spinning at 900 g atroom temperature for 20 min. (Beckman J6M/E centrifuge, JS-4.2 rotor).

2. Decant medium back into the cell-culture bottle (handle under sterileconditions the medium will be reused later), resuspend cells in 200-300mL MEM without serum (see Note 1), and transfer to a 1-L cell-culturebottle.

3. Infect cells with approx 10 PFU/cell of adenovirus from a high-titervirus preparation. Leave at 37° C. on a magnetic stirrer for 1 h. Dilutecells to approximately 4×10⁵ cells per mL in a large cell culture bottlewith the old MEM medium saved at step 2. Add fresh medium if necessary.4. Continue incubation at 37° C. for 20-24 h for preparation oflate-infected extracts. Additional protocols for infecting sickled cellswith various lytic viruses or tumor selective viruses are given inExample 11 and in Adenovirus Methods and Protocols WSM Wold, editor,Humana Press, Totowa, N.J. (1999) which is herein incorporated inentirety by reference.Preparation of the Hypoxia Responsive Element Promoter of the VEGF Gene

Cloning and Sequencing of the Mouse VEGF Promoter Region: The VEGFpromoter region is amplified by PCR using genomic DNA isolated frommouse liver, oligonucleotide primers synthesized on the basis of thepublished DNA sequence (GenBank accession number U41383), and LA Taq DNApolymerase (Takara Biomedicals, Osaka, Japan). The sense and antisenseprimers are −1215 (5′-TTTAGAAGATGAACCGTAAGC-CTAG-3′) and +315(5′-GATACCTCTTTCGTCTGCTGA-3′), respectively. The PCR conditions are 94°C. for 5 min followed by 30 cycles of 94° C. for 30 s, 68° C. for 3 min,and 72° C. for 7 min. The PCR product, which contained the 5′-flankingsequence encompassing the putative HRE site, the transcription startsite, and the 5′-untranslated region, is gel-purified and subcloned intoa TA cloning vector prepared from EcoRV-cut pBluescript KS−™(Stratagene, La Jolla, Calif.). Several independent clones aresequenced, and a clone is used for additional experiments. Deletion ofthe HRE site is obtained by digestion with BsaAl, a recognition site ofwhich resides in the middle of the HRE site.

Luciferase Reporter Plasmid Constructs and Luciferase Assays

The VEGF promoter sequence with or without the HRE site in pBluescriptKS− is excised by digestion with the appropriate restriction enzymes,gel-purified, and blunt-ended with T4 DNA polymerase, and the fragmentwas ligated into Smal-cut pGL2-Basic vector (Promega, Madison, Wis.),yielding plasmids pGLV(HRE)Luc or pGLV(AHRE)Luc, respectively. Theorientation of the insert is verified by restriction enzyme analysis.Transient transfection was carried out using Lipotectin (LifeTechnologies, Inc., Gaithersburg, Md.). As a control for transfectionefficiency, pRL-CMV vector (Promega) is cotransfected with testplasmids. pGL2-Control vector (Promega) was used as a positive control.Luciferase activity in cell extracts is assayed 48 h after transfectionaccording to the Dual-Luciferase reporter assay system protocols(Promega) using a luminometer (model TD-20/20; Turner Designs,Sunnyvale, Calif.).

Construction of Retroviral Vectors

Retroviral vector LXSN (provided by Dr. A. D. Miller, Fred HutchinsonCancer Research Center, Seattle, Wash.) is modified as follows to createa multicloning site. The retroviral vector is digested with EcoRI andXhoI and blunt-ended with T4 DNA polymerase. A SacI/KpnI fragment ofpBluescript SK− that is blunt-ended with T4 DNA polymerase is ligated tothis vector. This procedure yields retroviral vector LXSN(BA), which hasa multicloning site between the BstXl site and the Apal site ofpBluescript KS−. A retroviral vector harboring the VEGF promotersequence, HSV-TK gene or GFP gene, and SV40pA, all of which are locatedin a reverse orientation of LTR, is obtained as follows. A SV40pAfragment is prepared by digestion of pZeoSV (Invitrogen Corp., Carlsbad,Calif.) with Acc1 and BamHI. The fragment is gel-purified, blunt-endedwith T4 DNA polymerase, and ligated into Bxt/XI-cut and blunt-endedLXSN(BA), yielding a LXSN(BA)/pA vector. The VEGF promoter region withor without the HRE site in pBluescript KS− is excised with EcoRI and Sanand ligated into EcoRI/SalI-cut LXSN(BA)/pA, generating vectors LV(HRE)and LV(AHRE), respectively. The GFP or HSV-TK gene or any other genegiven in section 66 is cloned into the NotI site of these vectors viaNotI linkers. The orientation of the inserts is verified by restrictionenzyme analysis. The retroviral vectors generated by this procedure aretermed LV(HRE)GFP, LV(HRE)TK, and LV(AHRE)TK.

Plasmid Transfection and Retrovirus Infection

Al 1 cells are transfected with the: plasmids using Lipofection. Theretroviruses harboring LV(HRE)GFP or LV(HRE)TK are generated by a φ2packaging cell line. All cells were infected with the retroviruses inthe presence of 8 μg/ml polybrene (Aldrich Chemical Co., Inc.,Milwaukee, Wis.). The cells are cultured in the presence of 400 μg/mlG418 (Life Technologies, Inc., Grand Island, N.Y.) to select for cellsthat expressed vector-derived genes.

Evaluation of GFP Expression and Vascularity in Cryosections of Tumors

Cells: 2×10⁵) transfected with LV(HRE)GFP are s.c. injected into theflank of syngeneic C57BL/6 mice (Nippon SLC, Hamamatsu, Japan). Ten daysafter the injection, tumors are surgically removed and frozen in OCTcompound. Cryostat sections are fixed with cold acetone and washed withDPBS, and endogenous peroxidase is blocked with 3% hydrogen peroxide inmethanol for 10 min. The samples are washed three times with DPBS andincubated with DPBS containing 10% normal goat serum for 60 min to blocknonspecific binding sites. They are then incubated with rat antimouseCD31 antibody (PharMingen, San Diego, Calif.). Sections are washed withDPBS and incubated with TRITC-conjugated goat antirat IgG. Afterextensive washings with DPBS, samples are mounted in 50% glycerol inDPBS containing 1 mg/ml phenylenediamine. The fluorescence emitted fromGFP and TRITC is observed under a confocal laser microscope (Fluoview;Olympus, Tokyo, Japan).

Alternatively, cells are subjected to hypoxia for 16 h followed byexposure to GCV for 24 h in air, and the cell number was determined 2days after the treatment.

In Vivo Experiments. Cells (2.5×10⁵) retrovirally transduced withLV(HRE)TK or LV(HRE) are s.c. injected into 6-week-old female C57BL/6mice. Ten days after the inoculation, GCV diluted in DPBS is i.p.injected at a concentration of 30 mg/kg twice daily at 8-h intervals for5 days. DPBS alone is injected into control mice. Tumor growth ismonitored by caliper measurement of two diameters at right angles, andthe tumor mass is estimated from the equation volume=0.5×a×b², where aand b are the larger and smaller diameters, respectively.

Example 9

Construction of Adenovirus Vectors with Insertions for Superantigens

Superantigens are inserted into human adenoviruses (Ads) which are usedas live viral vector for expression of superantigens in mammalian cells.Adenoviruses vectors are exemplified here for insertion of thesuperantigen nucleotide. A mutant adenovirus with selectivity for P53deficient tumors is preferred such as ONYX-015. An efficient andflexible system for construction of adenoviral vector with insertions ordeletions in early regions 1 and 3 as described by Bett A J et al.,Proc. Natl. Acad. Sci. 91: 8802-8806 (1994) is given below. Similarprocedures insertion of the superantigen gene would be applied to theONYX-014 mutant.

Principle of Method:

Superantigen genes are inserted into adenoviral vectors using thefollowing principles and methods adapted from Bett, A J et al., Proc.Nat. Acad. Sci. 91: 8802-8806 (1994). Additional methods are given in abook titled Adenovirus Methods and Protocols Wold, WSM ed. Humana Press,Totowa, N.J. (1999) which is incorporated in entirety by reference.These methods involve insertion of the superantigen DNA either byoverlap recombination or by ligation insertion. The method exemplifiedbelow for insertion of SAg sequences uses the Ad5DNA virus but may beadapted to the dl1150 or ONYX-015 mutant or any other adenovirus. TheAd5 DNA sequences are cloned into bacterial plasmids. Deletions are madein the early region 1 and (3180 bp) and early region 3 (2690 or 3132 bp)and are combined in a single vector that have a capacity for inserts ofup to 8.3 kb, enough to accommodate the majority of cDNAs encodingproteins with regulatory elements. SAg genes are inserted into eitherearly region 1 or 3 or both and mutations or deletions are readilyintroduced into the viral genome.

SAg genes may be inserted into areas of the viral genome that have beeninactivated or deleted and considered to be non-essential to the lyticactivity of the virus or its ability to evade the host immune response.Both Ad and HSV carry genes that are not essential for viral replicationand these may be utilized for SAg insertion.

The first step is the construction of AdBHG, a virus that contains theAd5 genome with the deletion of E3 sequences from by 28,133 to 30,818and the insertion of a restriction enzyme site. The next step is thegeneration of a bacterial plasmid containing the entire AdBHG genome andsubsequent identification of infectious clones. Baby rat kidney (BRK)cells are infected with AdBHG under conditions that result in thegeneration of circular Ad5 genomes. At 48 h after infection, DNA isextracted from the infected Bark cells and used to transform E. coliHMS174 to ampicillin and tetracycline resistance. Plasmids with thecomplete AdBHG genome are selected. The final step is the generation ofthe pBHG10 by deleting the packaging signals in pBHG9 by partial BamHIdigestion and relegation. A Pac I restriction enzyme site unique to thisplasmid is present between Ad5 bp 28,133 and by 30,818 to permit foreigngene insertion. Because the packaging signal is deleted, pBHG10 isnon-infectious but cotransfections with plasmids that contain theleft-end Ad5 sequences including the packaging signal produce infectiousviral vectors with an efficiency comparable to that obtained with pJM17.

Use of the pBHGE3, pBHG10, or pBHG11 combined with the 3.2-kb deletionin E1 permits superantigen DNA inserts of ˜5.2, ˜7.9, and ˜8.3.respectively, into viral vectors. To test the capacity of the BHGsystem, a 7.8 kb consisting of the lacZ gene driven by the HCM promoter(E1-antiparallel orientation) and the SEB gene driven by the beta actinpromoter (E1-parallel orientation) are inserted into the 3.2-kb E1deletion. The 7.8-kb insert is constructed by inserting the 4.1-kb Xba Ifragment from the SEB gene containing the SEB gene driven by the betaactin promoter into the Xba I site in pHCMVsp1LacZ generating pH/acSEB.The isolate pH/acSEB expressed both lacZ and SEB at levels comparable tothose obtained with vectors containing single inserts.

The Method:

The first step involves the construction of AdBHG, a virus that containsthe Ad5 genome with the deletion of E3 sequences from by 28,133 to30,818 and the insertion of modified pBR322 at by 1339. AdBHG is made bycotransfection of 293 cells with purified viral DNA from Ad5 PacI,digested with Cia I and Xba I, and pWH3.

The next step involves the generation of a bacterial plasmid containingthe entire AdBHG genome and subsequent identification of infectiousclones. Baby rat kidney (BRK) cells are infected with AdBHG underconditions that result in the generation of circular Ad5 genomes. At 48h after infection DNA is extracted from the infected BRK cells and usedto transform E. coli HMS 174 to ampicillin and tetracycline resistance(Apr and Tetr, respectively). From two experiments, plasmid DNA from atotal of 10 colonies is screened by HindIII and BamHI/Sma I digestionand gel electrophoresis. Plasmids that appear to posses a complete AdBHGgenome are selected and all four are found to be infectious whentransfected into 293 cells.

The final step involves generation of pBHG10 by deleting the packagingsignals in pBHG9 by partial BamHI digestion and relegation. The left andright termini of the Ad5 genomes are covalently joined and a segment ofplasmid pBR322 is present between AdS bp 188 and 1339 to allowpropagation of pBHG10 in E. coli. A Pac I restriction enzyme site,unique in this plasmid, is present between AdS bp 28,133 and by 30,818to permit insertion of the superantigen genes. Because the packagingsignal is deleted, pBHG10 is noninfectious but cotransfections withplasmids that contain the left-end Ad5 sequences including the packagingsignal produce infectious viral vectors with an efficiency comparable tothat obtained with pJM17.

To generate two useful variants, pBHGE3 and pBHG11 are constructed fromthe original plasmid pBHG10. pBHGE3 permits construction of vectors withwt E3 sequences and pBHG11 increase the cloning capacity of resultingviral vectors. The 2.69-kb E3 deletion in pBHG10 removes the majorportions of all E3 mRNAs, the first E3 3′ splice acceptor site, and theL4 polyadenylylation site but leaves the E3 promoter, the 5′ initiationsite, the first E3 5′ splice donor site, and the E3b polyadenylylationsite intact. Viruses with the 2.69-kb E3 deletion have the same growthkinetics and progeny virus yields as wt virus. The 3.1-kb E3 deletion inpBHG11 removes two additional elements not removed by the 2.69-kb E3deletion: the first E3 5′ splice donor site and the E3bpolyadenylylation site. This deletion does not interfere with the openreading frame for pVIII or any of the L5 family of mRNAs. Virusescontaining the 3.1-kb deletion give wt progeny yields in infected 293cells.

To maximize the capacity of the BHG system and to facilitate theintroduction of inserts such as the SEB gene into the E1 region,plasmids containing a 3.2-kb deletion of E1 sequences and multiplerestriction sites for the insertion of foreign genes have beenconstructed. This deletion leaves intact the left ITR and packagingsignals and extends just past the Spi binding site of the protein IXpromoter. The promoter for transcription of the protein IX gene isrelatively simple, consisting of this Spi binding site and a TATA box.The Spi binding site is essential for expression of protein IX and it istherefore, reintroduced at a position 1 bp closer to the TATA box thanin the wt promoter. However, neither the original 3.2-kb E1 deletion northe deletion mutants containing the synthetic Spi site are significantlyaltered in protein IX expression, heat stability or final progeny yieldsof viruses with this deletion.

General Treatment Plan for Patients with the SAg-dl1150 Construct

SAg-dl1150 is administered intratumorally to patients with recurrent andrefractory cancers. The efficacy of SAg-dl1150 treatment is based on theinjected tumor(s) response. The clinical benefit of SAg-dl1150 isevaluated through quality-of-life assessment (EORTC instrument),Karnofsky performance score, and pain assessment. Survival andprogression-free survival intervals are recorded. Results are given inExample 23 (Table VIII).

SAg-dl1150 Dosages and Dosing Rationale: Patients are treated withSAg-dl1150 administered daily for 5 d at a dose of 10¹⁰ pfu per day.This is the highest dose administered daily for 5 d in the phase I studyand was shown to be safe (i.e., no dose-limiting toxicities).

Treatment with SAg-dl1150:

a. Dosing Regimen: For administration of each dose of, patients aretreated and observed in a properly equipped outpatient clinic. Thetarget tumor is injected with 10¹⁰ PFU of SAg-dl1150 daily over 5 d(i.e., a total dose 5×10¹⁰ PFU) (with day 1 being the first day ofSAg-dl1150 injection. Nontarget tumor(s) (where applicable) are injectedwith either diluent or SAg-dl1150 on the same days in identical fashionto the target tumor following the guidelines detailed in steps below.b. Target Tumor Masses: The dominant, symptom-causing tumor (if symptomsare present) is identified as the target tumor and is the only tumorinjected with SAg-dl1150 during the first two treatment cycles. Theidentification of the most symptomatic, problematic lesion is based onthe judgement of the Principal Investigator. Multinodular, butcontiguous tumors are treated and evaluated as a single lesion.c. Secondary, Nontarget Tumor Masses: If additional, smaller, accessiblelesions are present, these lesions are injected with diluent for thefirst two treatment cycles as described in step 3 below. Thereafter,treatments are divided between up to three separate lesions (i.e., theinitial two cycles are concentrated within the dominant lesion;thereafter, 6 wk after treatment initiation, two additional secondarylesions are injected). However, the total dose to the patient remainsthe same (i.e., the same total dose will be divided up between thetumors to be treated); the total volume in which the SAg-dl1150 issuspended will be increased based on the total tumor volume of thetumors to be treated. If a CR occurs in a treated lesion, injections canbe continued as outlined above with newly defined dominant and secondarylesions.d. Immediate Post treatment Monitoring of Patients: The patient's vitalsigns are taken 15 mm before each SAg-dl1150 injection. After eachinjection is completed, the patient will be observed in the clinic for aminimum of 30 mm. Vital signs are taken after 30 min±5 mm. If vitalsign(s) have changed by >15%, vital signs will be repeated every 30 mmuntil returning to within baseline 15% of baseline values. Following theobservation period, the patient is sent home or hospitalized overnightat the discretion of the investigator.

Example 10

Identification and Characterization of Streptococcal PyrogenicExotoxins, Staphylococcal Enterotoxins and SETs

SPEA Allelic Forms and Mutants. The method of preparation of SPEAallelic forms and mutants is carried out by the method of Kline J B etal., Infect. Immun. 64: 861-869 (1996).

Purification of SPEA from S. pyogenes. One-liter cultures of S. pyogenesRos (generous gift of D. L. Stevens, Idaho VA Medical Center) are grownin NCTC-135 medium (Gibco/BRL, Grand Island, N.Y.) supplemented withglucose (21). Toxin was partially purified from cell-free culturefiltrates by differential solubility in ethanol and acetate-bufferedsaline. Toxin which were precipitated four times were redissolved in 0.1M imidazole-acetic acid (pH 5.0) and applied to a QAE-Sephadex A-50(Pharmacia Fine Chemicals, Uppsala, Sweden) jacketed column. The toxinwas eluted as a single peak with a NaCl gradient as describedpreviously. Sodium dodecyl sulfate (SDS)-poly-acryl-amide gelelectrophoresis (PAGE) analysis of purified SPEA reveals a single bandwith the expected molecular mass of SPEA (25.8 kDa). The toxin isdialyzed against phosphate-buffered saline (PBS) and stored at −20° C.

Construction of pET15b-speal. 150 ng of plasmid pA2 containing the SPEAgene (kindly provided by J. J. Ferretti, Oklahoma City, Okla.) is usedas a template to amplify a 663-bp DNA fragment by PCR using primers

19b-Al (5′-CCCCATATGCAACAAGACCCCGAT-3′) and 19b-A2(5′-GGGGGATCCTTACTTGGTTGTTAG-3′).

These primers encode terminal BamHI and NdeI restriction sites,respectively. After digestion with BamHI and NdeI (Gibco/BRL), the DNAfragment, which encodes the mature protein without the leader peptide,is cloned into BamHI- and AMd-digested pET15b (Novagen, Madison, Wis.),producing the construct pET15b speA1. The complete nucleotide sequenceof the inserted fragment is confirmed by the dideoxy-chain terminationmethod. In E. coli BL21(DE3) (Novagen), this construct expresses afusion protein consisting of an N-terminal six-histidine-residue tag andSPEA1.

Generation of point mutations in SPEA. Site-directed mutagenesis ofSPEA1 is performed by using PCR with oligonucleotides containing thedesired nucleotide substitution. Briefly, 150 ng of pET15b-speA1, themutant oligonucleotide, and either primer 19b-A1 or primer 19b-A2 wereused to generate two SPEA fragments with complementary ends. A secondPCR is performed with the two overlapping SPEA fragments and flankingprimers 19b-A1 and 19b-A2 to generate the full-length mutated SPEA gene.This PCR product is then digested with BamHI and NdeI and inserted intopET15b as described above. The complete nucleotide sequences of bothstrands of each mutated SPEA are determined by the dideoxy-chaintermination method to ensure that only the single desired mutation waspresent.

Recombinant toxin nomenclature. Recombinant SPEA1 (rSPEA1) amino acidsubstitution mutants are named according to the original amino acid, itsposition in the mature toxin, and the resulting amino acid. For example,for rSPEA1-N₂0A, amino acid residue 20 was changed from asparagine toalanine. All mutant recombinant proteins generated contain single aminoacid substitutions except for rSPEA1-S51L, N55A and rSPEA1-C87S, C90S,which have two substitutions. rSPEA1 is the toxin encoded by SPEA1.rSPEA2 (also referred to as rSPEA1-G80S) is the toxin encoded

Expression and purification of rSPEA. Expression and purification of therecombinant toxins by using the pET expression vector is as described bymanufacturer (Novagen). In brief, E. coli BL21 (DE3) was transformedwith pET15-SPEA constructs for production of recombinant toxins. In thisbackground, SPEA is under the control of a T7 promoter, and the T7polymerase gene is on the E. coli chromosome under the control of anisopropylthio-D-galactopyranoside (IPTG)-inducible lac promoter.Cultures are grown to mid-exponential phase and induced to express toxinby the addition of 0.4 mM IPTG (Sigma Chemical Co., St Louis, Mo.).Cultures are grown for an additional 3 h after induction, harvested bycentrifugation, and disrupted by sonication. rSPEA preparations arepurified by metal chelation chromatography using His-Bind resin(Novagen). One hundred to 500 fg of toxin are digested with 1 μg ofthrombin (Novagen) for 16 h at room temperature. The toxin is thenpurified from the His-tag leader sequence by ultrafiltration with10,000-molecular-weight cutoff filters (MSI, Westboro, Mass.). In E.coli BL21(DE3) (Novagen), this construct expresses a fusion proteinconsisting of an N-terminal six-histidine-residue tag and SPEA.

Generation of polyclonal antisera recognizing SPEA. Female New ZealandWhite rabbits are by SPEA2. The toxin encoded by SPEA1, SPEA3, is alsotermed rSPEA1-V761. immunized subcutaneously with 50 μg of commerciallyavailable SPEA1 (Toxin Technologies, Sarasota, Fla.) in completeFreund's adjuvant (Gibco/BRL). Subsequent immunizations of 25 mg oftoxin are administered at week 3 and then every 2 weeks in incompleteFreund's adjuvant (Gibco/BRL). Sera were first collected at week 6.

Western blot (immunoblot) analysis of rSPEA. Each of the mutant toxinsand allelic forms is screened for instability by Western analysis.Toxins are analyzed by SDS-PAGE (12% acrylamide) and electroblotted tonitrocellulose. The nitrocellulose filters are incubated overnight inPBS supplemented with 5% low-fat dry milk and then stained withpolyclonal rabbit antiserum against SPEA1. Anti-SPEA antibody binding isdetected with horseradish peroxidase-labeled goat anti-rabbit antibody.Bands were visualized with 4-chloro-1-naph-thol (Sigma).

SDS-PAGE analysis. To look for the presence of disulfide bond formationbetween cysteine residues of rSPEA1,2-pg aliquots of purified toxins aremixed with gel running buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 0.1%bromophenol blue, 10% glycerol) with or without 2-mercaptoethanol (finalconcentration, 1%). The samples are then boiled for 5 min andelectrophoresed for 5 h at 40 mA on an SDS-12% polyacrylamide gelProtein bands were visualized by staining with Coomassie brilliant blueR250 (Bio-Rad, Melville, N.Y.).

Mitogenicity assays. Heparinized whole blood is obtained from healthydonors. Samples were fractionated on Ficoll-Paque (Pharmacia Biotech,Piscataway, N.J.), and the peripheral blood mononuclear cells (PBMCs)are harvested and washed three times in PBS. Then cells (10⁵) were addedto 96-well U-bottom plates in 200 μl of complete RPMI1640 supplementedwith 10% fetal calf serum (PCS). PBMCs are incubated for 72 h at 37° C.with various concentrations of rSPEA toxins under atmospheric conditionsof 5% CO₂; 1 uCi of [3H]thymidine (ICN Biochemical's, Costa Mesa,Calif.) is added to each well, and the cells are incubated for anadditional 24 h. Cells were harvested onto glass fiber filters, and[³H]thymidine uptake is quantitated by liquid scintillation counting.For each mutant toxin, PBMCs from at least three distinct donors areused.

Flow cytometry of PBMCs. PBMCs (10⁶) from healthy donors are incubatedwith toxins at a concentration of 1 μg/ml for 4 days. Cells wereharvested, washed three times with PBS, and applied to a FACScan flowcytometer (Becton Dickinson).

Cell lines. L-cell transfectants L66 (vector only) and L54.1(DQ(33/DQa2) are the generous gift of Robert Karr, Monsanto Company.Transfectants were maintained in suspension in petri dishes in Dulbeccomodified Eagle medium (DMEM) with 10% PCS, 2 mM L-glutamine, 100 U ofpenicillin per ml, 100 fig of streptomycin per ml, and 250 μg of theneomycin analog G418 per ml for selection. Before use, transfectants areexamined by fluorescence-activated cell sorting analysis withfluorescein isothiocyanate-labeled anti-HLA-DQ3 (KS13) to confirm theexpression and surface localization of the DQ molecule. Antibody KS13 isthe generous gift of Soldano Ferrone, New York Medical College,Valhalla, N.Y.

Radiolabeled rSPEA binding assays. rSPEA is iodinated by usingchloramine-T (Sigma). One hundred μg of toxin was incubated with 0.5 mCiof Na¹²⁵I and 5 μg (5 mg/ml) of chloramine-T in 100 μg of 100 mMTris-150 mM NaCl (pH 7.4) for 10 min. The reaction is terminated by theaddition of 20 μl (5 mg/ml) of sodium metabisulfite (Sigma). Labeledtoxin was separated from unincorporated radioactivity on a 1-ml SephadexG-25 column, which had been preequilibrated with PBS. The Kd of rSPEA-DQinteraction is determined by incubating 10⁶ L54.1 cells (expressingclass II MHC) with various concentrations of ml-rSPEA in a total volumeof 100 μg of DMEM-10% FCS-0.1% sodium azide. Nonspecific binding isestimated by incubating separate tubes with unlabeled competitor toxinat a concentration 100 times greater than that of labeled toxin. Cellsare incubated at 37° C. for 4 h with agitation every 20 min and thenpelleted through an oil gradient (80% dibutyl phthalate, 20% olive oil).Pellets are cut from the tubes, and cell-associated ¹²⁵I was measured ona gamma counter.

K determinations are evaluated in a similar fashion except thatadditional tubes containing various concentrations of 125I-rSPEA plusunlabeled mutant competitor are analyzed. Lineweaver-Burk plots of thereciprocal of toxin bound versus toxin free are used to determineinhibition constants.

Structure of SPEA. Predicted ribbon structure of SPEA was generated bythe Swiss Model Automated Protein Modelling Server, Glaxo Institute forMolecular Biology, Geneva, Switzerland. Primary amino acid sequence ofSPEA is modeled on the crystal structures of staphylococcal enterotoxinA (SEA) and staphylococcal enterotoxin E (SEE). Crystal coordinates forSEA and SEE are from the Brookhaven Database Crystal Coordinates and aredeposited by Swaminathan and Sax. Structure is viewed by using theRaswin Molecular Graphics Viewer software, version 2.4, 1994 (R. Sayte,Department of Computer Science, University of Edinburgh, Edinburgh,United Kingdom).

Isolation and Purification of SSA is by the method of Mollick J et al,J. Clin. Invest. 92: 710-719 (1993) and Reda K et al, Infect. Immun. 62:1867-1874 (1994)

Purification of SSA from S. pyogenes strain Weller. Because RDA has beenused to purify several staphylococcal enterotoxins, this material isuseful in identifying novel S. pyogenes superantigens. Concentratedculture supernatants from strain Weller are chromatographed on a RDAcolumn, the column was eluted with a phosphate step gradient, andfractions are tested for the presence of a class II-dependent T cellmitogen. We identify such an activity eluting between 60 and 150 mM P04,corresponding approximately to fraction numbers 8-55. This activityelutes in a broad peak and does not correspond to a detectable proteinpeak. Examination of an aliquot of the pooled activity by SDS-PAGE gelreveals many proteins, some in the 30-kD range. The pooled activefractions are fractionated from the RDA column by gel filtration (G-75)and anion exchange chromatography and active fractions from each columnare selected. The product from the final chromatography step consistspredominantly of three proteins. The proteins are blotted to a solidsupport and analyzed by NH2-terminal sequencing. The higher M_(r)protein is identified as SP and/or SPE-B. These two proteins are closelysimilar and are not distinguished based on the 29 amino acids sequenced.The lower M_(r) protein, −27 kD in size, yields a 59-amino acid NH2terminus that is not notably homologous to any previously characterizedprotein. The middle band (28 kD) displays an NH2 terminus strikinglysimilar to the NH2 termini of SEB, SEC, and SEC3 and dissimilar to Mprotein. The 28-kD molecule with the SEB-like NH2 terminus is designatedSSA.

Purification of SSA by Ab affinity chromatography. To determine whetheror not SSA is responsible for the superantigen activity, our efforts aredirected to its purification. An anti-peptide antiserum is raisedagainst the first 19 amino acids of SSA. To determine the ability of theanti-SSA Abs to bind native SSA, a concentrated streptococcalsupernatant from 16 liters is chromatographed on RDA in an effort toenrich for SSA. The RDA eluate is passed over the anti-SSAAb column andthe column eluted. Examination of the eluate by SDS-PAGE gel, and silverstain shows one prominent band at 28 kD, and two minor bands, one at −25kD and one at −12 kD. NH2-terminal sequencing of the 28-kD product showsthe SSA NH2 amino terminus. To determine whether the lower M_(r) specieswere contaminants or SSA degradation products, an identical sample issubjected to immunoblot analysis. Anti-SSA antibodies detect all threespecies shown in the silver stain gel, indicating that these lowermolecular weight bands are breakdown products of the 28-kD protein.Because the antibodies are directed against the NH2 terminus, theseproducts likely represent SSA molecules missing COOH terminal sections.

PCR amplification and cloning of the 5′ half of SSA from Weller genomicDNA. Nondegenerate, nonoverlapping oligonucleotides(SSA1,5′-AGTCAACCAGATC-CTACGCCAG AACAATTGAA-3′;SSA2,5′-AAATC-GAGTCAATTTAC GGAGTTATGGCC-3′) are designed on the basis ofthe SSA N-terminal protein sequence with a bias toward SEB codon usage.We hypothesized that SSA may retain homology to SEB in regions furtherdownstream from the 24 N-terminal residues, especially in regionsrelatively conserved among all known staphylococcal and streptococcalsuperantigens. In order to amplify SSA from Weller genomic DNA with PCR,we pair each SSA oligonucleotide with an oligonucleotide (SEB7, residues658 to 675) specific for a region in SEB immediately downstream of thedisulfide loop. Weller genomic DNA (200 ng) is combined with 50 pmoleach of sense (SSA1 or SSA2) and antisense (SEB7) primers, a 200 μlconcentration of each deoxynucleoside triphosphate, and 10 μl of 10×Pfupolymerase buffer 1 (Stratagene) in a total volume of 100 ul. Reactionmixtures are overlaid with 100 μL of mineral oil and denatured at 95° C.for 7 min before Pfu polymerase (2.5 units) (Stratagene) is added. PCRconditions were as follows: 1 min at 95° C., 2 min at 37° C., and 3 minat 72° C. for 25 cycles in a thermocycler (Perkin-Elmer Corp., Norwalk,Conn.). Combinations of SSA1 or SSA2 with SEB7 specifically amplifiedproducts of approximately 340 or 310 bp, respectively, from strainWeller genomic DNA, but not from strain Gall DNA, which does not produceSSA, PCR products were ligated to the pBluescript SK− vector to makepKR1 and pKR2, which are used to transform XL-1 Blue E. coli. Nucleotidesequence analysis of pKR1 and pKR2 inserts predicted amino acidsequences identical to that determined by N-terminal protein sequencingof native SSA from strain Weller S. pyogenes (39).

Subcloning and expression of recombinant SSA. The nucleotide sequenceencoding the mature form of SSA is PCR amplified from Weller genomic DNAwith flanking primers and digested with Hindi and Spel, which cut 46 bpupstream and 33 bp downstream, respectively, of the SSA open readingframe. This fragment was ligated to the pBluescript II KS expressionvector (Stratagene) to make pKR4. An XL-1 Blue E. coli strain carryingpKR4 was grown to an optical density at, 600 nm of 1.0 and induced toexpress SSA by the addition of IPTG(isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.1mM. After further incubation at 37° C. with shaking for 3 h, bacteriawere harvested by centrifugation and resuspended in TE, pH 8.0. Analiquot of cells was then mixed with an equal volume of SDS samplebuffer, and the whole cell lysate was analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and SSA immunoblot analysis. Lysates of E.coli strains carrying pMV7 or pBluescript, containing seb or no insertin the multicloning cassette, respectively, were processed in parallelas positive and negative controls. Uninduced whole cell lysates werealso examined.

Identification and Characterization SMEZ, SMEZ2, SPE-G, SPE-H and SPE-Jis given in Profit T et al. Exp. Med. 189: 89-101 (1999).

Identification of Novel SAgs. The novel SAgs are identified by searchingthe S. pyogenes Ml genome database at the University of Oklahoma withhighly conserved β5 and α4 regions of streptococcal and staphylococcalSAgs, using a TBlastN search program. The ORFs are defined bytranslating the DNA sequences around the matching regions and aligningthe protein sequences to known SAgs using the computer program Gap.Multiple alignments and dendrograms are performed with Lineup andPileup. We used the FAST A program for searching the SwissProt (AmosBairoch, Switzerland) and PIR (Protein Identification Resource) proteindatabases. The leader sequences of SPE-G and SPE-H are predicted usingthe SP Scan program. All computer programs are part of the GeneticsComputer Group package (version 8).

Cloning of smez, smez-2, spe-g, and spe-h. 50 ng of S. pyogenes Ml (ATCC700294) or S. pyogenes 2035 genomic DNA is used as a template to amplifythe smez DNA fragment and the smez-2 DNA fragment, respectively, by PCRusing the primers smez-fw (TGGGATCCTTA-GAAGTAGATAATA) and smez-rev(AAGAATTCTTAG-GAGTCAATTTC) and Taq Polymerase (Promega Corp.). Theprimers contain a terminal tag with the restriction enzyme recognitionsequences BamHI and EcoRI, respectively. The amplified DNA fragment,encoding the mature protein without the leader sequence is cloned into aT-tailed pBlueScript™ SKII vector (Stratagene).

Spe-g and spe-h are cloned by a similar approach, using the primers

spe-g-fw (CTGGATCCGATGAAAATTTAAAAGATT-TAA) and spe-g-rev(AAGAATTCGGGGGGAGAATAG), and spe-h-fw (TTGGATCCAATTCTTATAATACAACC) andspe-h-rev (AAAAGCTTTTAGCTGATTGACAC),respectively.The DNA sequences of the subcloned toxin genes are confirmed by thedideoxy chain termination method using a Licor automated DNA sequencer(model 4200). As the DNA sequences from the genomic database are allunedited raw data, three sub-clones of every cloning experiment areanalyzed to insure that no Taq polymerase-related mutations wereintroduced. The DNA sequence of the smez-2 gene has been annotated inEMBL/Genbank/DDBJ under accession number AF086626.

Expression and Purification of rSMEZ, rSMEZ-2, rSPE-G, and rSPE-H.Subcloned smez, smez-2, and spe-g fragments are cut from pBlueScript™SKH vectors, using restriction enzymes BamHI and EcoRI (GIBCO BRL), andcloned into pGEX-2T expression vectors (Pharmacia Biotech). Due to aninternal EcoRI restriction site within the spe-h gene, thepBlueScript:spe-h subclone is digested with BamHI and HindIII and thespe-h fragment is cloned into a modified pGEX-2T vector that contains aHindIII 3′ cloning site. rSMEZ, rSMEZ-2, and rSPE-H are expressed inEscherichia coli DH5a cells as glutathione-5-transferase (GST) fusionproteins. Cultures are grown at 37° C. and induced for 3-4 h afteradding 0.2 mM isopropyl-p-D-thiogalactopyranoside. GST-SPE-G fusionprotein is expressed in cells grown at 28° C. The GST fusion proteinsare purified on glutathione (GSH) agarose and the mature toxins arecleaved off from GST by trypsin digestion. All recombinant toxins,except rSMEZ, were further purified by two rounds of cation exchangechromatography using car-boxy methyl sepharose (Pharmacia Biotech). TheGST-SMEZ fusion protein is trypsin digested on the GSH-column and theflow-through containing the SMEZ is collected.

Gel Electrophoresis. All purified recombinant toxins are tested on a12.5% SDS-polyacrylamide gel according to Laemmli's procedure. Theisoelectric point of the recombinant toxins is determined by isoelectricfocusing on a 5.5% polyacrylamide gel using ampholine, pH 5-8 (PharmaciaBiotech). The gel is run for 90 min at 1 Watt constant power.

Toxin Proliferation Assay. Human PBLs are purified from blood of ahealthy donor by Histopaque Ficoll (Sigma Chemical Co.) fractionation.The PBLs are incubated in 96-well round-bottomed microtiter plates at10⁵ cells per well with RPMI-10 (RPMI with 10% PCS) containing varyingdilutions of recombinant toxins. The dilution series is performed in 1:5steps from a starting concentration of 10 ng/ml of toxin. After 3 d, 0.11 wCi[³H]thymidine is added to each well and cells are incubated foranother 24 h. Cells are harvested and counted on a scintillationcounter. Mouse leukocytes are obtained from spleens of five differentmouse strains (SJL, B10.M, B10/J, C3H, and BALB/c). Splenocytes arewashed in DMEM-10, counted in 5% acetic acid, and incubated onmicrotiter plates at 10⁵ cells per well with DMEM-10 and toxins asdescribed for human PBLs.

TCR Vβ Analysis. Vβ enrichment analysis is performed by anchoredmultiprimer amplification. Human PBLs are incubated with 20 pg/ml ofrecombinant toxin at 10⁶ cells/ml for 3 d. A twofold volume expansion ofthe culture followed with medium containing 20 ng/ml IL-2. After another24 h, stimulated and resting cells are harvested and RNA is preparedusing Trizol reagent (GIBCO BRL). A 500 bp βchain DNA probe is obtainedby anchored multiprimer PCR, radiolabeled, and hybridized to individualVβ5 and a Cβ DNA region dot-blotted on a Nylon membrane. The membrane isanalyzed on a Storm PhosphorImager using ImageQuant software (MolecularDynamics). Individual Vβs are expressed as a percentage of all the Vβsdetermined by hybridization to the Cβ probe.

Jurkat Cell Assay. Jurkat cells (a human T cell line) and LG-2.cells (ahuman B lymphoblastoid cell line, homozygous for HLA-DR1) are harvestedin log phase and resuspended in RPMI-10. 100 ul of the cell suspension,containing 10⁵ Jurkat cells and 2×10⁴ LG-2 cells are mixed with 100 μlof varying dilutions of recombinant toxins on 96-well plates. Afterincubating overnight at 37° C., 100-^1 aliquots are transferred onto afresh plate and 100 μl (10⁴) of Sel cells (IL-2-dependent murine T cellline) per well are added. After incubating for 24 h, 0.1 1μCi[³H]thymidine is added to each well and cells are incubated foranother 24 h. Cells were harvested and counted on a scintillationcounter. As a control, a dilution series of IL-2 is incubated with Selcells.

Computer-aided Modelling of Protein Structures. Protein structures ofSMEZ2, SPE-G and SPE-H are created on a Silicon Graphics computer usingInsight11/Homology software (Biosym Technologies). The SAgs SEA, SEB,and SPE-C are used as reference proteins to determine structurallyconserved regions (SCRs). Coordinate files for SEA (1ESF), SEB (1SEB),and SPE-C (1AN8) are downloaded from the Brookhaven Protein Database.The primary amino acid sequences of the reference proteins and SMEZ-2,SPE-G, and SPE-H, respectively, are aligned, and coordinates fromsuperimposed SCRs are assigned to the model proteins. The loop regionsbetween the SCRs are generated by random choice. MolScript software isused for displaying the computer-generated images.

Methods of isolation and characterization of SPEC is carried out by themethods of Li P L et al., J. Exp. Med. 186: 375-383 (1997)

Toxin Purification. All toxins are expressed from the pGEX vector inEscherichia coli as glutathione S transferase (GST) fusion proteins andpurified by glutathione chromatography. Mature toxins are cleaved fromGST by trypsin digestion and purified by two rounds of cation exchangechromatography. The first round uses carboxymethyl sepharose and thesecond on a POROS HS (Perceptive Systems, Cambridge, Mass.) HPLC column.All toxins are resistant to trypsin digestion except SEB which has asingle cleavage site in the disulphide loop region. This does not affectSEB activity.

Toxin Proliferation Assays. Human peripheral blood lymphocytes arepurified by Ficoll-Hypaque and incubated for 3 d at 106 cells/ml induplicate in 96-well microtiter plates in media containing varyingdilutions of recombinant toxins. 0.1 fid [3H]thymidine is added to eachwell, and cells were incubated a further 24 h. Plates are harvested andcounted on a scintillation counter.

TCR Vβ Analysis. These are performed using the reverse dot-blotprocedure. In brief, human peripheral blood lymphocytes are incubatedwith 1 ng/ml of recombinant toxins for 3 d. The cultures are expandedtwofold with medium containing 20 ng/ml IL-2. Cells are harvested at 4 dand RNA made by standard procedures. TCR β-chain messenger is reversetranscribed using a set of primers specific for a conserved region inall β-chain genes. Amplification of a 500-bp Vβ probe is accomplished byan anchor primer to the 5′ end of the β-chain primers plus a singleCβregion primer. This probe is radiolabeled and reverse blotted tofilters containing individual β-chain genes. Relative changes inindividual β-chain mRNA are compared to unactivated PBL.

Anti-TCR mAb FACS Staining. Activated T cells are incubated for 1 h onice with 25 ml of anti-TCR BV2 (MPB2/C11; a gift from A. W. Boylston,University of Leeds, Leeds, UK), anti-BV551 (LC4; a gift from R. Levy,Stanford University Medical School, Stanford, Calif.), anti-BV5S3(42/1C1; a gift from A. W. Boylston, University of Leeds, Leeds, UK),anti-BV8.1 (C305; a gift from A. Weiss, University of California, SanFrancisco, Calif.), and anti-BV12S (S511; a gift from D. Posnett,Cornell University Medical College, NY). Washed cells are then incubatedwith 1 ml FITC goat anti-mouse (Becton Dickinson) and incubated on icefor a further 30 min. After washing, cells are analyzed on a FACSCAN®.

Zinc Blots. Recombinant toxins (10 (μg) are incubated in triplicate with10 μg EDTA followed by 100 μg 65ZnCl (New England Nuclear, Boston,Mass.) in 20 mM Tris, pH 8.0, 10 mM MgCl2, 0.15 M NaCl made zinc free byaddition of chelex resin (Sigma Chemical Co.). Samples are then dotblotted to nitrocellulose filters using a 96-well dot-blot apparatus.Filters are washed briefly three times with zinc-free buffer, and thenautoradiographed. Spots are cut out and counted on gamma counter(Packard Instrs., Meriden Conn.) to quantify 65Zn bound to each toxin.

Nondenaturing SDS Electrophoresis. Toxin samples are incubated instandard Laemmli reducing sample buffer (containing 1% SDS and 10 mMdithiothreitol), and then resolved as normal of a 12.5% acrylamide gel.For denaturing conditions, samples are heated to 100° C. for 2 minbefore loading. To prevent dimers from dissociating during running, thepower is maintained below 2 W (20 mA and 100 V). Some reduced samplesare treated with 20 mM iodoacetic acid, pH 7.0, before loading. EDTA isadded to some samples at 10 mM. incubation at 37° C. as the percentageof LG-2 cells in aggregates, and is determined by light microscopy.

Western Blotting of S. pyogenes Strain 2035-de-rived SPE-C. S. pyogenesstrain 2035 is grown under anaerobic conditions in brain heart fusionmedium at 37° C. for 24 h without shaking. Supernatant proteins areconcentrated by sequential (NH₄)₂SO₄ precipitations, with cuts of <40%,40-60% 60-80%, and 80% saturation, and resuspended in 50 mM Tris-1 mMEDTA, pH 7:4, at 500-1,000 times their original concentration.Recombinant SPE-C or 10 ul of the 60-80% (NH₄)₂SO₄-precipitable fractionsupernatant are combined with an equal volume of nonreducing 2% SDSsample buffer and separated by 0.1% SDS-12% PAGE. Denatured samples areheated (95° C.) for 2 min before analysis. Fractions are dialyzedextensively against 25 mM Tris-50 mM NaCl-1 mM EDTA, pH 7.4, to removesalt. After separation by SDS-PAGE, proteins are transferred to anitrocellulose filter (Hybond-C; Amersham Corp., Arlington Heights,111.) in an electroblotting apparatus 8.5-150 mM glycine-10% methanol).The filter is blocked in PBS-0.05% Tween-5% nonfat dried milkpowder-0.1%) normal rabbit serum and stained with 1:6,000peroxidase-labeled affinity-purified rabbit and-SPE-C immunoglobulin.The peroxidase conjugate is detected on radiographic film bychemiluminescence (ECL; Amersham Corp.) according to the manufacturer'sinstructions.

Size Exclusion Chromatography. Recombinant SPE-C. (2 mg/ml) is dialyzedat 4° C. overnight in 20 mM BisTris-Tris, pH 6.0 or 9.0. 20 μl samplesare diluted into 100 μl 50 mM BisTris, pH 6.0, 7.0, 8.0, or 9.0/0.1 MNaCl and incubated for 1 h at room temperature before separation at 1ml/min (±0.05 ml/min) on a Superose12 (Pharmacia) high resolution HPLCcolumn attached to a Biocad Sprint (Perceptive Systems) preequilibratedwith the respective incubation buffer. Trace chromatograms monitoringAbs_(280 nm), pH, and conductivity are all recorded directly andsubsequently analyzed for retention times, peak integration, and peakassignment using the on-line Biocad software. Traces are grouped andprinted using the stacked trace mode which automatically aligns eachtrace to the injection point

Identification and Characterization of the Staphylococcal enterotoxinsSEG, SEH, SEI, SEJ, SEK, SEL, SEM is carried out by the method ofJarraud S J Clinical Microbiology (1999).

Strains. S MJB1316 (a gift from Sibyl Munson, University of Wisconsin,Madison, Wis.), an RN450 derivative that contains the cloned seg gene onthe staphylococcal expression vector pRN5548, is used as SEG positivecontrol. The following S. aureus strains were used to check thespecificity of PCR amplification: FDA-S6 (ATCC 13566 (sea+ seb+)),FRI-137 (ATCC 19095 (sec+, seg+, seh+, sei+)), FRI-1151 m (sed+),FRI-326 (ATCC 27664 (see+)), FRI-569 (ATCC 51811 (seh+)), FRI-1169(tst+), TC-7 (eta+, seg+, sei+), and TC-146 (etb+ seg+ sei+). Twohundred thirty S. aureus clinical isolates are collected. They areisolated from 58 patients with S. aureus infection (arthritis, skininfection, pneumonia, or infective endocarditis), 102 patients withacute toxemia (TSS, SSF, or SSSS), and 70 asymptomatic nasal carriers.All strains are collected from hospitals located throughout France andare identified as S. aureus by their ability to coagulate citratedrabbit plasma (bioMerieux, Marcy-I'Etoile, France) and to produce aclumping factor (Staphyslide Test; bioMerieux). Escherichia coli TGI isused for plasmid amplification and genetic manipulations.

DNA amplification and sequencing DNA is extracted from A900322 culturesand used as a template for amplification with primers sei-1 and seg-2.Primers wsei and wseg are designed following identification of suitablehybridization sites in the sei and seg genes and were compatible withthe Clontech Genome Walker kit (Ozyme; Montigny-Le Bretonneux, France),which is suitable for cloning unknown DNA sequences adjacent to a knownsequence. This kit is used, according to the supplier's instructions, toidentify sei and seg flanking regions using primers hindlll and wsei ona HindIII chromosomal digest for the amplification of the sei-upstreamregion; and primers hpa1 and wseg on an Hpa1 chromosomal digest for theamplification of the SEG-downstream region. PCR products are analyzed byelectrophoresis through 0.8% agarose gels (Sigma, St. Louis, Mo.),purified using the High Pure PCR Product Purification kit (BoehringerMannheim, Meylan, France), and sequenced (Genome Express, Grenoble,France). Sequences are compiled, analyzed, and compared using Blast(http://www.ncbi.nlm.nih.gov/BLAST), Gene-Jokey, and ClustalX software(European Bioinformatics Institute, Cambridge, U.K.,http://www.ebi.ac.uk).

Toxin-gene detection. Sequences specific for sea-e, seg-i, tst, eta, andetb, encoding SEA-E, SEG-I, TSST-1, ETA, and ETB, respectively, aredetected by PCR. DNA from clinical isolates is extracted from culturesand used as a template for amplification with the primers described inTable 1 (Eurogentec, Seraing, Belgium). Table 1 of primers used isgiven-in Jarraud et al., J. Clin. Micro. (1999). Amplification of gyrAis used as a control to confirm the quality of each DNA extract and theabsence of PCR inhibitors. All PCR products are analyzed byelectrophoresis through 1% agarose gels (Sigma).

Detection of bacterial RNA by RT-PCR. Total RNA is extracted fromstaphylococcal cultures by using RNeasy spin columns (Qiagen,Courtaboeuf, France). cDNA is synthesized using Ready-To-Go RT-PCR beads(Pharmacia Biotech, Orsay, France) by incubating 0.1 μg of total RNAwith the following pairs of primers (primer 5′, sel3), (sel-4, sel-5),(sel 1, sel2), (invsel2, invsem1), (semi, invsei1), (sei1, sei2),(invsei2, ψent2), (ψent1, invsek 1), (sek1, sek2), (invsek2, invseg1),(seg1, seg2), (invseg2, primer 3′). The reaction mixtures are incubatedwith each primer pair described above, at 42° C. for 30 min for reversetranscription, followed by 30 cycles of amplification (1-mindenaturation at 94° C. 1-min annealing at 55° C., and 1-min extension at72° C.). The RT-PCR products are then analyzed by electrophoresisthrough 1% agarose gel. RNA extracts are tested for DNA contamination bypreincubating the reaction mixtures at 95° C. for 10 min to inactivatereverse transcriptase before the RT-PCR.

Production and purification of recombinant enterotoxins. Primers aredesigned following identification of suitable hybridization sites insel, sem, sei, sek, and seg. The 5′ primers are chosen within the codingsequence of each gene, omitting the region predicted to encode thesignal peptide, as determined by hydrophobicity analysis according toKyte and Doolitttle with GeneJockey software and SignalP VI.1 World WideWeb Prediction Server (www.cbs.d-tu.dk/services/SignalP/); the 3′primers are chosen to overlap the stop codon of each gene. A restrictionsite is included in each primer. DNA is extracted from A900322 orMJB1316 and used as a template for PCR amplification. PCR products andplasmid DNA are prepared using the Qiagen plasmid kit. PCR fragmentswere digested with EcoRI and PstI (Boehringer Mannheim) and ligated (T4DNAligase; Boehringer Mannheim) with the pMAL-c2 expression vector fromNew England Biolabs (Ozyme) digested with the same restriction enzymes.The resulting plasmids are transformed into E. coli TG 1. The integrityof the ORF of each construct is verified by DNA sequencing of thejunction between pMAL-c2 and the different inserts. The fusion proteinsare purified from cell lysates of transfected E. coli by affinitychromatography on an amylose column according to the supplier'sinstructions (New England Biolabs).

T cell proliferation assays. PBL from healthy donors are cultured in24-well plates (10⁶ cells/well) in RPM 11640 medium supplemented with 8%pooled human serum and 10 fig/ml recombinant staphylococcal toxin. rIL-2(50 IU/ml) is added on day 5. When necessary, T cell cultures arediluted in IL-2-supplemented medium until TCR analysis. For controls Tcells from the same donors that are stimulated with 0.5 μg/ml Phaseolusvulgaris leucoagglutimn (PHAL) (Sigma) are used.

Flow cytometry. The following mAb (mAb; specificity indicated inbrackets) are used for flow cytometry: E2.2E7.2 (Vβ2), LE89 (Vβ3),IMMU157 (Vβ5.1), 3D11 (Vβ5.3), CRI304.3 (Vβ6.2), 3G5D15 (Vβ7), 56C5.2(V8.1/8.2), FIN9 (Vβ9), C21 (Vβ11), S511 (Vβ12), IMMU1222 (Vβ13.1) JIJ74(Vβ13.6), CAS1.1.13 (Vβ14), Tamaya1.2 (Vβ16), E17.5F3 (Vβ17), BA62.6(Vβ18), ELL1.4 (Vβ20), IG125 (Vβ21.3), IMMU546 (Vβ22), and HUT78.1(Vβ23). These mAb, and CD4- and CD5-specific mAb, is purchased fromBeckman/Couker/Immunotech (Marseille, France). Cells are phenotyped byindirect immunofluorescence, as described previously. Briefly, cells areincubated with unconjugated mAb for 30 mm at room temperature, thenwashed and incubated with FITC-conjugated rabbit anti-mouse Ig antiserum(BioAtlantic, Nantes, France) for 30 min on ice. After washing, cellsare analyzed by flow cytometry on a FACScan apparatus (Becton Dickinson,Mountain View, Calif.) using the LYSYS II software package on aFACstation.

Immunoscope analysis. Total RNA is extracted using the Tnzol reagent(Life Technologies, Gaithersburg, Md.). TCR (3-chain-specific primersare as described previously, and reverse transcription, PCRamplification, and run-off steps are performed as reported previously.Fluorescent DNA products are loaded on a sequencing gel and analyzedwith the Immunoscope software.

Identification of the SEG and SEI flanking regions. When this work wasinitiated, the coding regions of only seg and sei were available, andthe two genes were known to be in tandem orientation, separated by a1.9-kb DNA fragment in S. aureus strain A900322. A 3.2-kb fragment isthus amplified by PCR with primers sei1 and seg2 and was then sequenced.The intergenic 1.9-kb DNA sequence contains three open reading frames(ORF1, 2, and 3) of 399, 327, and 777 bp, respectively. Comparison ofthe deduced amino acid sequences of these ORFs with translated sequencesfrom GenBank showed that the putative proteins corresponding to theseORFs had substantial sequence similarities to known SEs: ORF1 exhibitedhomology to the N-terminal region of SEB; ORF2 to the C-terminal regionof SEC; and ORF3 to SEA. The PCR “walking” strategy is chosen toidentify the seg and sei flanking regions. The use of primers wsei andhindlll on Hindlll digests amplifies and allows sequencing of the 3.2 kbof DNA upstream of sei. Analysis of this sequence showed two significantORFs (ORF4 and ORF5) of 783 and 720 bp, respectively. ORF4 exhibitedhomology with SEJ, and ORF5 with SEI. The use of primers wseg and hpa1on Hpa1 digests amplified a 0.8-kb fragment downstream of seg. Sequenceanalysis of this fragment reveals no other significant ORFs. Theconcatenated sequence of seg-sei-intergenic, -upstream and -downstreamregions is validated by sequencing a 6.189-kb PCR fragment encompassingthe whole region. Although sei in strain A900322 is 100% homologous withthe sequence deposited in GenBank (accession number AF064774), seg instrain A900322 showed one mutation, corresponding to a Leu→Prosubstitution at position 29. This new variant is designated SEGL29P.ORFs 1-5 are homologous but not identical with any known enterotoxinshence they corresponded to new enterotoxins. However, ORF1 and 2 are atleast 50% shorter than any of the known enterotoxins. ORF-1 possesses asatisfactory Shine-Dalgarno (SD) sequence (TGGAGT-N7-AUG, consensusAGGAGG-N6/10-AUG) but, in comparison with SEB, to which it is highlyrelated, shows a large deletion of its 3′ end, which corresponds to aregion that is essential for biological (superantigenic) activity. ORF2has neither an SD sequence nor a signal peptide, and resembles anN-terminal-truncated SEC. Accordingly, ORF1 and 2 are designated ψent1and ψ2, respectively, meaning they represent pseudogenes with no likelybiological function. In contrast, ORFs 3, 4, and 5 have sizes consistentwith active enterotoxin-like molecules. ORF5 possesses a satisfactory SDsequence and translation start site, whereas ORF3 and ORF4 have anadequate SD sequence in front of a nonca-nonical, although suitable,translation start site (ATT) coding the thiamine. Thus. ORF3, ORF4, andORFS are designated sek, sel, and sem, respectively. Thus, the 6301-bpDNA region identified contains seg and set plus three potentialenterotoxin genes (sek, sel, and sem) and two pseudogenes (ψent1,ψent2), all in the same orientation. We designated this region egc for“enterotoxin gene cluster.” With the exception of plasmid pIB485, whichcontains SED and SEJ in opposite orientations separated by 895nucleotides, and the staphylococcal pathogenicity island, which containstst and ent separated by 10.234 kb, no such gene cluster organizationhas been previously described for enterotoxin genes.

Transcriptional analysis. To investigate whether this seg transcript waspolycistronic, i.e., encoded one or more of the ORFs identified in egc,c-DNA is generated from strain A900322 total RNA by reversetranscription and amplified by PCR using primer pairs located withineach gene and bracketing adjacent genes. Abundant RT-PCR products (B toK) of the expected size are obtained using the corresponding primerpairs. In contrast, no RT-PCR product A (primer 5′, seI3) nor L (primerinvseg2 and primer 3′) is obtained. These results suggest that the sevengenes and pseudogenes composing egc are cotranscribed, and that the 5′and 3′ ends of the transcript must be close to the beginning of sel andto the end of seg, respectively. Sequence analysis reveal putative −10and −35 promoter sequences (TTGTCT-N15-TAATTT-N134-ATT) upstream of thesel start codon. The 3′ end may lie at an inverted repeat at position6018-6067, which is a potential transcription termination signal, 5830nucleotides downstream of the putative transcription start site. Theseresults suggest that egc is an operon.

Superantigen activity. The association of related genes that arecotranscribed suggested that the resulting peptides might havecomplementary effects on the host's immune response. One hypothesis isthat gene recombination created new variants of toxins differing bytheir superantigen profiles. Purified recombinant SEL, SEM, SEI, SEK,and SEGL29P expressed in E. coli are studied for their ability to induceselective expansion of T cells bearing particular TCR Vβ regions inshort-term PBL culture. As shown in Tables III and IV, recombinant SELSEM, SEI, and SEK consistently induces selective expansion of distinctsets of Vβ subpopulations. By contrast. SEGL29P fails to triggerexpansion of any of the 23 β subsets. The sum of results obtained witheach of these recombinant toxins globally corresponds to the selectiveexpansion of Vβ subpopulations induced by crude supernatant ofstaphylococcal culture of strains that harbored egc (data not shown).This suggests that the maltose-binding protein portion of the fusiontoxins do not significantly influence the Vβ specificity of thesesuperantigens. To investigate whether the L29P mutation could explainthe lack of superantigen activity, a rSEG with an L29 codon isconstructed from S. aureus strain MJB1316 (which contains the cloned segon a plasmid) and then expressed in E. coli, and the superantigenactivity of this toxin is tested. SEGL29. induces selective expansion ofVβ14 and, to a lesser extent, Vβ13.6, OT cells. The L29P mutation thusaccounts for the complete loss of superantigen activity. Computermodeling of the two-dimensional structure of the wild-type and mutatedproteins reveals no major conformational differences between the twoproteins. It is likely that L29 is located at a position crucial forproper superantigen/MHC II interaction. In addition to the selectiveexpansion of TCR Vβ subsets observed with the different toxins, flowcytometry reveals preferential expansion of CD4 T cells in SEI and SEMcultures. By contrast, the CD4/CD8 ratios in SEK-, SEL-, andSEG-stimulated T cell lines are close to those in fresh PBL. Thisphenomenon, which is observed with cells from several donors, reflects avariable contribution of the CD4 coreceptor to the T cell activationprocess, depending on the affinity of the TCR for the superantigen/MHCcomplex. To document the TCR Vβ composition of superantigen-stimulated Tcell lines and the clonal diversity of the expanded TCR Vβ subsets, thesize distribution of PCR-amplified TCR β-chain junctional products isstudied using the Immunoscope technique. Results of this molecularanalysis are in good overall agreement with those obtained by flowcytometry, as similar dominant TCR Vβ subsets are identified with thetwo approaches. Additionally, Immunoscope analysis shows that thecomplementarity-determining region 3 size distribution of TCR β-chainjunctional transcripts within expanded Vβ subsets is pseudogaussian inall superantigen-stimulated cultures, reflecting a high level ofpolyclonality. This is further confirmed by sequence analysis of TCR βjunctional transcripts derived from some expanded TCR Vβ subsets. Takentogether, these TCR repertoire studies confirm the superantigenic natureof the new toxins identified in this study.

Example 11

Construction of Adenovirus Vectors with Insertions for Superantigens

Superantigens are inserted into human adenoviruses (Ads) which are usedas live viral vector for expression of superantigens in mammalian cells.Adenoviruses vectors are exemplified here for insertion of thesuperantigen nucleotide. A mutant adenovirus with selectivity for P53deficient tumors is preferred such as ONYX-015. An efficient andflexible system for construction of adenoviral vector with insertions ordeletions in early regions 1 and 3 as described by Bett A J et al.,Proc. Natl. Acad. Sci. 91: 8802-8806 (1994) is given below. Similarprocedures insertion of the superantigen gene would be applied to theONYX-014 mutant.

Principle of Method: Superantigen genes are inserted into adenoviralvectors using the following principles and methods adapted from Bett, AJ et al., Proc. Nat. Acad. Sci. 91: 8802-8806 (1994). Additional methodsare given in a book titled Adenovirus Methods and Protocols Wold, WSMed. Humana Press, Totowa, N.J. (1999) which is incorporated in entiretyby reference. These methods involve insertion of the superantigen DNAeither by overlap recombination or by ligation insertion. The methodexemplified below for insertion of SAg sequences uses the Ad5DNA virusbut may be adapted to the dll 150 or ONYX-015 mutant or any otheradenovirus. The Ad5 DNA sequences are cloned into bacterial plasmids.Deletions are made in the early region 1 and (3180 bp) and early region3 (2690 or 3132 bp) and are combined in a single vector that have acapacity for inserts of up to 8.3 kb, enough to accommodate the majorityof cDNAs encoding proteins with regulatory elements. SAg genes are,inserted into either early region 1 or 3 or both and mutations ordeletions are readily introduced into the viral genome. SAg genes may beinserted into areas of the viral genome that have been inactivated ordeleted and considered to be non-essential to the lytic activity of thevirus or its ability to evade the host immune response. Both Ad and HSVcarry genes that are not essential for viral replication and these maybe utilized for SAg insertion.

The first step is the construction of AdBHG, a virus that contains theAd5 genome with the deletion of E3 sequences from by 28,133 to 30,818and the insertion of a restriction enzyme site. The next step is thegeneration of a bacterial plasmid containing the entire AdBHG genome andsubsequent identification of infectious clones. Baby rat kidney (BRK)cells are infected with AdBHG under conditions that result in thegeneration of circular Ad5 genomes. At 48 h after infection, DNA isextracted from the infected BRk cells and used to transform E. coliHMS174 to ampicillin and tetracycline resistance. Plasmids with thecomplete AdBHG genome are selected. The final step is the generation ofthe pBHG1O by deleting the packaging signals in pBHG9 by partial BamHIdigestion and relegation. A Pac I restriction enzyme site unique to thisplasmid is present between Ad5 bp 28,133 and by 30,818 to permit foreigngene insertion. Because the packaging signal is deleted, pBHG10 isnoninfectious but cotransfections with plasmids that contain theleft-end Ad5 sequences including the packaging signal produce infectiousviral vectors with an efficiency comparable to that obtained with pJM17.

Use of the pBHGE3, pBHG10, or pBHGl1 combined with the 3.2-kb deletionin E1 permits superantigen DNA inserts of ˜5.2, −7.9, and −8.3.respectively, into viral vectors. To test the capacity of the BHGsystem, a 7.8 kb consisting of the lacZ gene driven by the HCM promoter(E1-antiparallel orientation) and the SEB gene driven by the beta actinpromoter (E1-parallel orientation) are inserted into the 3.2-kb E1deletion. The 7.8-kb insert is constructed by inserting the 4.1-kbXba Ifragment from the SEB gene containing the SEB gene driven by the betaactin promoter into the Xba I site in pHCMVsplZacZ generating pH/acSEB.The isolate pH/acSEB expressed both lacZ and SEB at levels comparable tothose obtained with vectors containing single inserts.

The Method: The first step involves the construction of AdBHG, a virusthat contains the Ad5 genome with the deletion of E3 sequences from by28,133 to 30,818 and the insertion of modified pBR322 at by 1339. AdBHGis made by cotransfection of 293 cells with purified viral DNA from Ad5PacI, digested with Cia I and Xba I, and pWH3.

The next step involves the generation of a bacterial plasmid containingthe entire AdBHG genome and subsequent identification of infectiousclones. Baby rat kidney (BRK) cells are infected with AdBHG underconditions that result in the generation of circular Ad5 genomes. At 48h after infection DNA is extracted from the infected BRK cells and usedto transform E. coli HMS 174 to ampicillin and tetracycline resistance(Apr and Tetr, respectively). From two experiments, plasmid DNA from atotal of 104 colonies is screened by Hindlll and BamHI/Sma I digestionand gel electrophoresis. Plasmids that appear to posses a complete AdBHGgenome are selected and all four are found to be infectious whentransfected into 293 cells.

The final step involves generation of pBHG 10 by deleting the packagingsignals in pBHG9 by partial BamHI digestion and relegation. The left andright termini of the Ad5 genomes are covalently joined and a segment ofplasmid pBR322 is present between AdS bp 188 and 1339 to allowpropagation of pBHG10 in E. coli. A Pac I restriction enzyme site,unique in this plasmid, is present between AdS bp 28,133 and by 30,818to permit insertion of the superantigen genes. Because the packagingsignal is deleted, pBHG10 is noninfectious but cotransfections withplasmids that contain the left-end Ad5 sequences including the packagingsignal produce infectious viral vectors with an efficiency comparable tothat obtained with pJM17.

To generate two useful variants, pBHGE3 and pBHGl1 are constructed fromthe original plasmid pBHG10. pBHGE3 permits construction of vectors withwt E3 sequences and pBHGl 1 increase the cloning capacity of resultingviral vectors. The 2.69-kb E3 deletion in pBHGlO removes the majorportions of all E3 mRNAs, the first E3 3′ splice acceptor site, and theL4 polyadenylylation site but leaves the E3 promoter, the 5′ initiationsite, the first E3 5′ splice donor site, and the E3b polyadenylylationsite intact. Viruses with the 2.69-kb E3 deletion have the same growthkinetics and progeny virus yields as wt virus. The 3.1-kb E3 deletion inpBHGl 1 removes two additional elements not removed by the 2.69-kb E3deletion: the first E3 5′ splice donor site and the E3bpolyadenylylation site. This deletion does not interfere with the openreading frame for pVIII or any of the L5 family of mRNAs. Virusescontaining the 3.1-kb deletion give wt progeny yields in infected 293cells. [01654] To maximize the capacity of the BHG system and tofacilitate the introduction of inserts such as the SEB gene into the Elregion, plasmids containing a 3.2-kb deletion of E1 sequences andmultiple restriction sites for the insertion of foreign genes have beenconstructed. This deletion leaves intact the left ITR and packagingsignals and extends just past the Spi binding site of the protein IXpromoter. The promoter for transcription of the protein IX gene isrelatively simple, consisting of this Spi binding site and a TATA box.The Spi binding site is essential for expression of protein IX and it istherefore, reintroduced at a position 1 bp closer to the TATA box thanin the wt promoter. However, neither the original 3.2-kb E1 deletion northe deletion mutants containing the synthetic Spi site are significantlyaltered in protein IX expression, heat stability or final progeny yieldsof viruses with this deletion.

ADDITIONAL DOCUMENTS INCORPORATED BY REFERENCE

This application incorporates by reference the following patents andcurrently pending patent applications that disclose inventions of thepresent inventor alone or with co-inventors.

Application Ser. No., Pat. No. or Date of filing, issuance PublicationNo. Title or publication WO 91/10680 Tumor Killing Effects ofEnterotoxins and published 25 Jul. 1991 Related Cpds USSN 07/891,718Tumor Killing Effects of Enterotoxins and filed 01 Jun. 1992. RelatedCpds U.S. Pat. No. 5,728,388 Method of Cancer Treatment issued Mar. 17,1998. USSN 08/491,746 Method of Cancer Treatment filed 19 Jun. 1995.USSN 08/898,903 Method of Cancer Treatment filed 23 Jul. 1997. USSN08/896,933 Tumor Killing Effects of Enterotoxins and filed 18 Jul. 1997.Related Cpds USSN 60/085,506 Compositions and Methods for Treatmentfiled 05 May 1998. of Cancer USSN 60/094,952 Compositions and Methodsfor Treatment filed 31 Jul. 1998. of Cancer USSN 60/033,172Superantigen-Based Meth and filed 17 Dec. 1996. Compositions forTreatment of Cancer USSN 60/044,074 Superantigen-Based Meth and filed 17Apr. 1997. Compositions for Treatment of Cancer USSN 09/061,334 TumorCells with Increased filed 17 Apr. 1998. Immunogenicity and Uses ThereofUSSN 09/311,581 Compositions and Meth for Treating filed 14 May 1999.Neoplastic Disease USSN 60/173,371 Compositions and Meth for Treatingfiled 28 Dec. 1999 Neoplastic Disease, USSN 05/208,128 Compositions andMeth for Treating filed 31 May 2000 Neoplastic Disease USSN 09/650,884Compositions and Meth for Treating filed 28 Dec. 2000 Neoplastic DiseaseUSSN 09/870,759 Compositions and Meth for Treatment of filed 5 May 2001Neoplastic Disease USSN 60/389,366 Compositions and Meth for Treatmentof filed 15 Jun. 2002 Neoplastic Disease USSN 60/406750 IntrathecalSuperantigens to Treat filed 29 Aug. 2002 Malignant Fluid AccumulationUSSN 60/406,697 Compositions and Meth for Treatment of filed 28 Aug.2002 Neoplastic Diseases USSN. 60/378,988 Compositions and Meth forTreatment of Filed 8 May 2002 Neoplastic Diseases USSN 09/751,708Compositions and Meth for Treatment of Filed 28 Dec. 2000 NeoplasticDiseases

Moreover, all references cited herein are incorporated by reference,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

What is claimed is:
 1. A method of delivering a therapeutic agent to asolid tumor characterized by hypoxia, acidosis and hypertonicitycomprising loading the therapeutic agent into mature human sickle redblood cells or human nucleated sickle cell progenitor cells whichexpress at least one hemoglobin S allele and administering said maturehuman sickle red blood cells or human nucleated sickle cell progenitorcells, into which said therapeutic agent is loaded, into the bloodcirculation of a human patient having said solid tumor wherein saidmature sickle red blood cells or nucleated sickle cell progenitor cellsaccumulate in said solid tumor in order to treat said solid tumor. 2.The method according to claim 1, wherein the therapeutic agent loadedinto the mature human sickle red blood cells or human nucleated sicklecell progenitor cells is an anti-tumor virus, toxin, siRNA, drug orprodrug.
 3. The method according to claim 1, wherein said at least onehemoglobin S allele expressed in said mature human sickle red bloodcells or human nucleated sickle cell progenitor cells is linked to asecond hemoglobin allele selected from the group consisting ofhemoglobin S, hemoglobin A, hemoglobin C and β thalassemia hemoglobin.4. The method according to claim 2, wherein said antitumor virus isselected from the group consisting of herpes simplex, adenovirus,vaccinia, Newcastle Disease virus, reovirus and autonomous parvovirus.5. The method of claim 2, wherein said toxin consists of: (i) a wildtype staphylococcal enterotoxin or wild type streptococcal pyrogenicexotoxin protein which wild type protein has the biological activity ofstimulating T cell mitogenesis via a T cell receptor vβ region; (ii) abiologically active variant or fragment of a wild type staphylococcalenterotoxin or streptococcal pyrogenic exotoxin, which variant orfragment: (a) has the biological activity of stimulating T cellmitogenesis via a T cell receptor vβ region and (b) has sequencehomology characterized as a z value exceeding 13 when the sequence ofthe variant or fragment is compared to the sequence of a wild typestaphylococcal enterotoxin or a wild type streptococcal pyrogenicexotoxin, determined by FASTA analysis using gap penalties of −12 and−2, Blosum 50 matrix and Swiss-PROT or PIR database; or (iii) abiologically active fusion protein comprising: (A) said variant, (B)said wild type staphylococcal enterotoxin, (C) said wild typestreptococcal pyrogenic exotoxin, or (D) said fragment, operably linkedto a peptide or polypeptide fusion partner.
 6. The method of claim 2,wherein the toxin is a staphylococcal enterotoxin selected from thegroup consisting of SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEK,SEL, SEM, SSA, and TSST-1 or wherein the toxin is a Streptococcalpyrogenic exotoxin selected from the group consisting of SPEA, SPEB,SPEC, SPEC, SPEJ, SPEH SME-Z, and SME-Z₂.
 7. The method of claim 2,wherein said toxin is fused to a fusion partner selected from the groupconsisting of an antibody or antibody fragment specific for an antigenor receptor expressed on a tumor cell or tumor vasculature and anArg-Gly-Asp (RGD)-containing polypeptide.
 8. The method of claim 5,wherein said peptide or polypeptide fusion partner is a coaguligandselected from the group consisting of inactivated factor VIII, tissuefactor, thrombin, factor V/Va, factor VHI/VIIIa, factor IX/IXa, factorX/Xa, factor X1/XIa, factor XH/XIIa, factor XIII/XIIIa, factor Xactivator and factor V activator.
 9. The method of claim 2, wherein saiddrug is a chemotherapeutic agent selected from the group consisting of aheavy metal, an antimetabolite, an anthracycline, a vinca alkaloid, ananti-tubulin agent, an antibiotic and an alkylating agent.
 10. Themethod of claim 2, wherein said drug is a chemotherapeutic agentselected from the group consisting of docetaxel, paclitaxel, taxotere,cisplatin, doxorubicin, vinorelbine, gemcitabine, camptothecin,dactinomycin, mitomycin, caminomycin, daunomycin, tamoxifen,vincristine, gemcitabine, vinblastine, etoposide, 5-fluorouracil,cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate,actinomycin-D, mitomycin C, aminopterin, and combretastatin.
 11. Themethod of claim 1, wherein said administration of said mature humansickle red blood cells or human nucleated sickle progenitor cells is byinfusion or injection, intravenously or intraarterially.
 12. The methodaccording to claim 2, wherein said toxin is selected from the groupconsisting of a pertussis toxin, a pseudomonas toxin, a verotoxin, aricin, a C. perfringens exotoxin, a granzyme B, a perforin, a complementmembrane attack complex, a staphylococcal alpha hemolysin, anEscherichia coli hemolysin, a staphylococcal erythrogenic toxin, astreptococcal erythrogenic toxin, a staphylococcal coagulase, astaphylococcal beta hemolysin, and a Clostridia perfringens toxin. 13.The method of claim 2, wherein said toxin is a mutant or variant of awild type toxin which has the biological activity of the wild type toxinand has sequence homology characterized as a z value exceeding 13 whenthe sequence of the variant or said fragment is compared to the sequenceof a wild type toxin, determined by FASTA analysis using gap penaltiesof −12 and −2, Blosum 50 matrix and Swiss-PROT or PIR database or abiologically active fusion protein comprising said mutant or variantfused to a peptide or polypeptide fusion partner.
 14. The method ofclaim 1, wherein said therapeutic agent is selected from the groupconsisting of an enzyme, an apolipoprotein, an angiostatin, astaphylococcal protein A, a chemokine, a chemoattractant, a cytokine, aheat shock protein and a thrombospondin.
 15. The method of claim 14,wherein the enzyme is selected from the group consisting of acarbohydrate modifying enzyme, a galactosyltransferase, a staphylococcalhyaluronidase, a streptococcal hyaluronidase and a staphylococcalcoagulase.
 16. The method of claim 7, wherein said receptor expressed onsaid tumor cell or said tumor vasculature is an epithelial growthfactor.
 17. The method of claim 1, wherein therapeutic agent is loadedinto the mature human sickle red blood cells or the human nucleatedsickle progenitor cells by endocytosis, electroporation or physicalencapsulation.
 18. The method of claim 1, wherein said therapeutic agentis loaded into the human nucleated sickle progenitor cells bytransfection.
 19. The method of claim 18, wherein said therapeutic agentcomprises recombinant DNA, cDNA or genomic DNA.
 20. The method of claim19, wherein said recombinant DNA, cDNA or genomic DNA is incorporatedinto a vector, an autonomously replicating plasmid, a virus or thegenomic DNA of a prokaryote or eukaryote.
 21. The method of claim 20,wherein said virus is selected from the group consisting of aretrovirus, adenovirus, or a herpes virus.
 22. The method of claim 18,wherein said therapeutic agent loaded into said nucleated sickleprogenitor cells by transfection is operably linked to a hypoxiasensitive global operator and promoter.
 23. The method of claim 2,wherein said toxin is fused to EGF or inactivated factor VIII.